Host-Derived Lactic Acid Disrupts IFN Clinical Efficacy via Antiviral Inhibition and Proinflammatory Amplification

Host-Derived Lactic Acid Disrupts IFN Clinical Efficacy via Antiviral Inhibition and Proinflammatory Amplification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Host-Derived Lactic Acid Disrupts IFN Clinical Efficacy via Antiviral Inhibition and Proinflammatory Amplification Zhixin Liu, Mingfu Tian, Siwei Chen, Ju Huang, Xuan Wang, Moran Li, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8905265/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Emergent, sudden-onset, and highly prevalent viral pathogens severely threaten public health. Current vaccines/antiviral drugs are limited by narrow tropism, susceptibility to escape mutations, and prolonged development, highlighting an urgent need for broad-spectrum agents. Type I interferons exhibit potent broad-spectrum antiviral activity in preclinical studies but suffer from critical clinical limitations: narrow intervention windows for acute infections, suboptimal efficacy, and notable adverse reactions. The discrepancy between their robust preclinical and limited clinical performance remains mechanistically unclear. Herein, integrating clinical samples and multi-level infection models, we demonstrate that IFNs exert antiviral effects only when administered pre-infection. Once infection is established, IFNs are ineffective yet induce prominent adverse effects, with host-derived lactic acid (LAC) as the key mediator: it promotes viral immune evasion, impairs IFN therapeutic efficacy, triggers inflammatory storms, and elicits adverse reactions. Mechanistically, LAC suppresses IFN activity via membrane receptor-mediated SIRT1 upregulation and synergizes with IFNs to hyperactivate NF-κB, initiating cytokine storms and forming an "antiviral failure-inflammatory amplification" feedback loop. Based on this mechanism, we developed a combinatorial therapy of IFNs plus an FDA-approved lactate dehydrogenase inhibitor. This regimen reverses LAC-mediated IFN suppression, mitigates inflammation, and achieves dual "antiviral + anti-inflammatory" benefits. Notably, it retains robust efficacy even in late-stage infections, overcoming IFN monotherapy drawbacks and addressing the core bottleneck restricting IFN clinical application. Our study identifies LAC as a pivotal target for broad-spectrum antiviral development and provides a potential strategy to combat emerging viral pandemics. Health sciences/Pathogenesis/Infection Biological sciences/Microbiology/Virology/Virus–host interactions Lactic Acid Type I interferons Antiviral Activity Proinflammatory Effects Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Driven by accelerating globalization and ongoing ecological alterations, viral infections have emerged as a major disruptive threat to global public health. Frequent cross-species transmission and global viral pandemics not only pose a life-threatening risk to human health, leading to substantial morbidity and mortality, but also trigger systemic public health crises with profound socioeconomic impacts( 1 , 2 ). Although vaccination and antiviral interventions offer effective approaches to contain known epidemic viruses, their inherent limitations—narrow viral tropism, susceptibility to escape mutants, and a development cycle that lags behind viral evolution—render them ineffective against emerging and unknown viral pathogens. Thus, the development of broad-spectrum antiviral agents that are independent of specific viral types and possess pan-inhibitory activity has become a core imperative in global public health. This holds important strategic value for constructing a pre-emptive defense barrier against emerging viral infections and safeguarding global public health security. Within the host’s immune regulatory network against viral infections, interferons (IFNs) hold a central role as the primary known broad-spectrum antiviral factors ( 3 , 4 ), exerting potent inhibitory effects on diverse viruses regardless of viral species. Extensive preclinical research studies have demonstrated robust antiviral activity of IFNs in both in vitro cellular systems and in vivo animal models. For instance, pretreatment with recombinant IFN-α reduces influenza virus titers by 10²–10³ fold in MDCK cells( 5 ), through targeting viral RNA replication and protein translation. Conversely, IFN-α/β receptor-deficient mice succumb to lethal infection within 3–5 days following low-dose challenge with vesicular stomatitis virus (VSV) or Semliki Forest virus (SFV), with tissue viral loads elevated by 10³–10⁴ fold relative to wild-type littermates. In contrast, wild-type mice rapidly induce IFN responses to clear the virus within 72 hours, with 100% survival( 6 ). During the COVID-19 pandemic, IFNs similarly exhibited significant antiviral potential, with multiple in vitro and in vivo studies confirming their efficacy in suppressing viral replication and mitigating host tissue damage. Collectively, these findings establish IFNs as indispensable effector molecules of innate immunity, thereby providing a theoretical and experimental foundation for the development of IFN-based broad-spectrum antiviral strategies—addressing the unmet global public health need highlighted earlier. However, a profound discrepancy persists between the potent broad-spectrum antiviral potential of IFNs in preclinical studies and their limited clinical utility( 7 ). Currently, their clinical indications are highly restricted—approved only for a handful of conditions including chronic hepatitis B (CHB), chronic hepatitis C (CHC), and hairy cell leukemia( 8 – 11 ). Even for HCV, IFNs are not first-line therapy and are reserved for specific patient cohorts meeting strict eligibility criteria( 12 ).More critically, for acute viral infections with higher incidence and broader public health impact (e.g., acute influenza, COVID-19, dengue fever), IFNs are not recommended for routine use; instead, their application is hampered by suboptimal efficacy( 13 ) and elevated risk of adverse events( 14 – 16 ). For instance, while IFN-α treatment shortens viral shedding by 1–2 days in acute influenza patients, it fails to substantially alleviate clinical manifestations such as fever and cough, and may induce adverse reactions including headache, fatigue, and myelosuppression( 17 ). In early acute COVID-19, despite in vitro and animal studies demonstrating viral replication inhibition by IFN-α/λ, multicenter randomized controlled trials (RCTs) show no improvement in hospitalization or severe disease progression rates among mild cases. For severe cases, IFN therapy may even exacerbate lung injury via excessive inflammatory activation, worsening clinical outcomes( 18 ).The core regulatory mechanisms underlying this stark divide between IFN's preclinical promise and clinical performance remain incompletely defined, highlighting an urgent need for systematic mechanistic investigations to facilitate the rational clinical deployment of IFNs. Emerging evidence indicates that the clinical limitations of IFNs stem from multifaceted factors. Animal studies have confirmed that the protective effects of IFNs against infections such as SARS-CoV and MERS-CoV are strictly dependent on prophylactic administration or early post-infection intervention( 19 ). However, due to limitations in viral detection technologies and patient presentation timelines, early clinical intervention is rarely achievable, resulting in missed optimal treatment windows. Mechanistically, IFNs exert antiviral effects primarily by activating the JAK-STAT pathway—inducing antiviral effector molecule expression to suppress viral replication, while regulating the activation and differentiation of dendritic cells and T cells to constrain excessive inflammation( 20 ). By the time acute viral infection patients seek clinical care, however, viruses have already completed initial replication, immune responses are initiated, and some patients have entered a phase of hyperinflammation. At this stage, IFN therapy not only fails to exert antiviral effects but also its forced activation of the inflammatory responses and worsening disease severity( 21 ). Although backed by some clinical studies, the causes of IFN therapeutic failure and adverse effects remain unclear, with no effective countermeasures available. Elucidating the mechanisms underlying IFN therapeutic failure and adverse effects in late-stage treatment is critical for optimizing IFN usage strategies, expanding clinical applications, and developing broad-spectrum antiviral agents, thus offering novel therapeutic directions for addressing current and future emerging viral pandemics. To address this key scientific question, we integrated analyses of clinical samples, and cell/animal models of multi-viral infections, demonstrating that host-derived lactic acid (LAC) produced following established viral infection is the central driver of IFN therapeutic failure. Lactate not only directly suppresses IFN antiviral activity but also induces cytokine storms in the presence of IFNs, exerting dual effects that exacerbate host damage and undermine clinical outcomes. Mechanistically, lactic acid directly inhibits IFN antiviral signaling transduction via activating cell membrane receptors and downstream SIRT1 signaling. Concurrently, in synergy with IFNs, lactic acid potently activates the NF-κB pathway to induce inflammatory storms, forming a double negative feedback loop characterized by "antiviral failure and enhanced inflammation". Based on this mechanism, we developed a specific combination therapy of IFN plus the lactic acid dehydrogenase inhibitor stiripentol—an FDA-approved antiepileptic drug. This regimen effectively reduces host lactic acid production, reverses LAC-mediated antiviral suppression and pro-inflammatory effects, and achieves the dual benefit of "antiviral activity + inflammation control" with IFN therapy. Successfully overcoming some clinical limitations of IFNs, this combined therapy improves late-stage treatment efficacy and reduces side effects, providing a novel strategy for the development of broad-spectrum antiviral agents and offering new therapeutic prospects for combating current and future viral pandemics. Results 3.1 Interferon treatment loses efficacy and induces a proinflammatory response in the context of established viral infection. As a central effector molecule of the innate immune network, interferons (IFNs) are well-recognized key broad-spectrum antiviral agents. Their potent antiviral activity, independent of viral species, has been fully validated both in vitro cell systems and in vivo murine models. However, a stark contrast exists between the robust antiviral potential of IFNs in preclinical studies and their severe clinical limitations. Currently, clinical indications of IFNs are extremely narrow—approved only for a few disorders such as CHB and CHC, and often not as first-line therapy options. For acute viral infections with high incidence and broad impact—including influenza and COVID-19—IFNs are not recommended for routine use due to limited efficacy and elevated risk of adverse events. To date, the mechanisms underlying IFN therapeutic failure and adverse effects remain elusive, and no effective solutions exist to address this clinical dilemma, which greatly hindering the full realization of their clinical potential and the development of broad-spectrum antiviral strategies. To systematically investigate the core mechanisms limiting the clinical utility of IFNs and define the critical time window for IFN-α intervention, we established multiple virus-infected cell models (Fig. 1 A). By varying the timing of treatment and validating across multiple viruses and cell types, we elucidated its mode of action and universality. We selected two representative viruses: one is Influenza virus, a respiratory-transmitted pathogen responsible for multiple global pandemics with substantial mortality. Its seasonal epidemics, high mutation rate, limited vaccine protection, and narrow clinical window for antiviral drugs make it a major public health concern. The other is Vesicular stomatitis virus (VSV), a widely used laboratory model virus with broad tropism and distant phylogenetic relationship to influenza virus, facilitating validation of mechanism’s generality. Combined results showed that pretreatment with 2 nM IFN-α 2 h prior to infection effectively suppressed viral replication, whereas IFN-α antiviral activity was significantly attenuated once infection was established (Fig. 1 ; Figure S1 ). Specifically, pre-infection IFN-α pretreatment (-2 h) markedly inhibited the replication of influenza A virus (H1N1) and VSV in human non-immune A549 cells, reducing viral RNA and protein expression levels. This effect was consistently validated in immune cells (murine peritoneal macrophages, PM) and other primary cells (murine lung fibroblasts, MLF) (Fig. 1 B and 1 C; Figure S1 A-C). In contrast, IFN-α administration following viral infection significantly impaired antiviral efficacy with even early post-infection treatment (4 h) resulted in reduced viral activity (Fig. 1 B and 1 C; Figure S1 A-C). Furthermore, considering the pro-inflammatory potential of IFNs, we investigated this aspect. Results indicated that IFN-α pretreatment significantly suppressed virus-induced secretion of proinflammatory cytokines ( IL-1β, IL-6, TNF-α ) (Fig. 1 D, Figure S1 D). However, after infection is established, IFN-α therapy not only lacked anti-inflammatory effects but also significantly exacerbated inflammatory responses at later stages, particularly in influenza virus-infected cells (Fig. 1 D; Figure S1 D). These findings clearly demonstrate that IFN antiviral efficacy is strictly time-dependent: optimal antiviral effects are only achieved when administered pre-infection. Following established viral infection, therapeutic efficacy is drastically diminished and accompanied by proinflammatory adverse effects—an observation independent of virus and cell type, with substantial generality. To further validate the in vivo relevance of in vitro findings, we recapitulated the experimental design in murine models (Fig. 1 E). Mice were infected with different viruses and treated with IFN-α either pre-infection or post-infection, with systematic analysis of viral replication and pathological damage across infected tissues. Results demonstrated that compared to pre-infection pretreatment, IFN-α administration following infection significantly reduced antiviral potency. Even treatment initiated one day post-infection showed compromised efficacy, with later treatments correlating with weaker antiviral activity (Fig. 1 F; Figure S1 E and S1F). In contrast, IFN-α pretreatment effectively suppressed viral replication in mice (Fig. 1 F; Figure S1 E and S1F). More importantly, compared to infection alone, post-infection IFN-α therapy significantly exacerbated inflammatory responses in the liver and lung tissues of mice, inducing more severe histopathological damage and inflammatory cell infiltration (Fig. 1 G and H; Figure S2A-C). Conversely, IFN-α pretreatment effectively inhibited viral replication, significantly attenuating inflammatory responses and histopathological damage (Fig. 1 E-H; Figure S1 D-F; Figure S2). This pattern was consistently observed across different viral infection models and tissue types, confirming its in vivo generality. In summary, our study demonstrates that IFN-α administration prior to viral infection fully exerts its antiviral effects while significantly reducing inflammatory responses and virus-induced histopathological damage. In contrast, IFN-α therapy initiated after viral infection is established not only results in drastically diminished antiviral activity but also exacerbates inflammatory responses and tissue injury. Notably, this phenomenon exhibits broad generality across different viral infection models, cell types, and in vivo tissues. Fundamentally, the prophylactic intervention models of "pre-infection pretreatment" or "concomitant administration" used in preclinical studies are not clinically feasible, as prophylactic pre-infection use is rarely achievable, and optimal timing is difficult to determine. Clinical interventions for diagnosed patients typically occur after infection is established, where treatment induces a double negative effect of "antiviral failure and enhanced side effects"—this constitutes the core reason why preclinical IFN efficacy cannot be fully recapitulated in clinical practice. 3.2 Established Viral Infection Suppresses IFN-α Signaling Transduction The antiviral effects of IFN-α are primarily mediated by its binding to interferon receptors, leading to activation of the downstream JAK-STAT signaling pathway and subsequent induction the expression of core antiviral factors including MX1( 22 ) and OAS1( 23 ). In this study, we observed a significant attenuation of IFN-α antiviral activity following established viral infection. We therefore hypothesized that impaired JAK-STAT signaling transduction following infection is the key mechanism underlying IFN-α therapeutic failure. To test this hypothesis, we administered IFN-α at two critical time points (12 h and 24 h) following established influenza or VSV infection (Fig. 2 A and 2 C), using direct IFN-α treatment (without viral infection) as a control. Results showed that IFN-α failed to effectively activate the JAK-STAT signaling pathway following established viral infection in both non-immune cells (A549, MLF) and immune cells (PM). In contrast, IFN-α treatment efficiently activated this pathway and its downstream effectors in normal, uninfected cells. Specifically, following infection with different viruses, the phosphorylation of STAT1—a key upstream transducer in the IFN signaling pathway—was significantly suppressed (Fig. 2 B and 2 D; Figure S4A). Concurrently, the expression levels of downstream core interferon-stimulated genes ( ISGs ), including ISG15, MX1, IFIT1 , and OAS1 , were markedly downregulated (Figure S3A and S3B; Figure S4B and S4C). These findings demonstrate that impaired JAK-STAT signaling transduction following established viral infection is the core mechanism of IFN-α antiviral failure. This regulatory effect was consistent across different viruses and cell types, indicating broad generality. To further validate these findings in vivo , we established a murine model of H1N1 influenza virus infection. Mice were treated with IFN-α (200 ng per mouse) post-infection, and lung tissues were collected for analysis (Fig. 2 E). In vivo results were fully consistent with in vitro observations: compared to IFN-α treatment in uninfected mice, IFN-α treatment following viral infection failed to effectively activate the JAK-STAT signaling pathway in mouse lung tissues. Expression of key downstream effector genes ( ISG15, MX1, IFIT1 , and OAS1 ) was significantly reduced, ultimately limiting IFN-α antiviral efficacy (Fig. 2 F). These in vivo data confirm that established viral infection specifically suppresses IFN-α-mediated JAK-STAT signaling transduction and downstream effector expression. In summary, through integrated in vitro and in vivo experiments, this study demonstrates that IFN-α fails to effectively activate the JAK-STAT signaling pathway following established viral infection—this constitutes the key reason for its its diminished antiviral potency. Importantly, this mechanism exhibits generality across different cell types and viral infection models. 3.3 Lactic Acid Mediates the Suppression of IFN-α Antiviral Signaling After Viral Infection Prior studies have confirmed that IFN-α-mediated JAK-STAT signaling is significantly suppressed following established viral infection, and this inhibitory effect is conserved across multiple cell types and viral infection models. This suggests the existence of a universal regulatory mechanism that transcends infection contexts and cellular backgrounds to impair the antiviral function of the IFN pathway. To elucidate the molecular mechanism underlying this universal suppression, we performed transcriptomic sequencing on VSV infected PM cells. In the present study, an in-depth analysis was conducted on the most significantly upregulated genes following infection. Combined with the focused mining of differentially expressed genes (DEGs) and the results of TOP 20 pathway enrichment analysis, our findings revealed that: after viral infection, in addition to the significant activation of immune-related pathways, glucose metabolism-related pathways were also significantly upregulated, among which the glycolysis pathway-related genes exhibited the most prominent changes in expression, (Fig. 3 A-C), indicating that the glycolytic pathway is prominent activated by viruses. To verify the causal link between aberrant glucose metabolism and JAK-STAT pathway suppression, we pretreated A549 and PM cells with key upstream/downstream glycolytic inhibitors (2-deoxy-D-glucose [2-DG], lactate dehydrogenase inhibitor Oxamate, and pyruvate transport inhibitor UK-5099), followed by IFN-α stimulation during viral infection (Fig. 3 D). Results demonstrated that inhibiting early glycolytic steps and lactate dehydrogenase effectively restored IFN-α-mediated JAK-STAT signaling activation (Fig. 3 E-F), indicating that the impaired IFN-α responsiveness in the late phase of viral infection is closely associated with lactate dehydrogenase, a key downstream glycolytic enzyme. Lactate dehydrogenase (LDH) primarily catalyzes the metabolism of pyruvate to lactate, whereas inhibition of LDH restores interferon (IFN) activity (Fig. 3 E-F). Building on this observation, we further investigated the role of lactate in suppressing IFN function. We found that intracellular lactic acid levels increased progressively over time following viral infection, with a significant elevation observed as early as the acute phase of infection (Fig. 3 G)—consistent with the early impairment of IFN-mediated antiviral activity following infection (Fig. 1 ; Figure S1 ). Furthermore, in murine models, serum LAC levels were significantly elevated in virus-infected mice (Fig. 3 H). More importantly, analysis of clinical samples confirmed the pathological relevance of this phenomenon: COVID-19 patients exhibited significantly higher LAC levels relatived to healthy individuals (physiological normal concentration of lactate, 0.5–1.6 mmol/L) (Fig. 3 I, left). Dynamic monitoring of LAC levels in influenza patients revealed a marked upward trend 3 days after positive infection detection, with no significant change at initial positive infection detection (Fig. 3 I, right)—consistent with the progressive elevation of lactic acid induced by viral infection. Collectively, these results from cellular, animal, and clinical studies consistently demonstrate that viral infection induces a significant increase in host LAC levels, with clear clinical relevance. Based on these observations, we propose a central hypothesis: Lactate derived from glucose metabolism is the key effector inhibiting IFN-α-mediated JAK-STAT signaling activation during viral infection. Notably, lactate exerts concentration-dependent effects (physiological vs. supraphysiological). Elevated lactate (exceeding physiological levels) was detected in our cellular models, murine specimens, and clinical samples post-infection, with concentrations reaching up to 10 mmol/L in subsets of patients and mice (Fig. 3 G-I). To better reflect clinical practice—where interferon therapy is typically initiated after infection establishment, when patients already exhibit elevated lactate (Fig. 3 I)—we used supraphysiological lactate concentrations in our treatment regimens, and interferon treatment was given after lactic acid treatment. A549 cells, PMs, and MLFs were pretreated with 10 mM lactic acid for 24 h, followed by IFN-α stimulation (Fig. 3 I). Results demonstrated that lactic acid directly and significantly suppressed IFN-α-mediated JAK-STAT signaling activation. Specifically, in the presence of lactic acid, IFN-α-induced phosphorylation of STAT1 (a key upstream molecule in the pathway) and the expression of downstream core antiviral genes ( ISG15 , MX1 , IFIT1 , OAS1 ) were significantly inhibited across multiple cell types (Fig. 3 J; Figure S5A and S5B). These findings clearly confirm that elevated host lactic acid induced by viral infection directly inhibits IFN signaling transduction, thereby impairing host antiviral capacity. To verify the in vivo generality of this mechanism, we reproduced the experiments in murine models (Fig. 3 K). Results were fully consistent with in vitro observations: lactic acid significantly suppressed IFN-α-mediated JAK-STAT signaling activation in mice (Fig. 3 L; Figure S5C). Compared to direct IFN-α treatment, the presence of lactic acid reduced STAT1 phosphorylation levels and significantly inhibited the expression of downstream antiviral genes ( ISG15 , MX1 , IFIT1 , OAS1 ) in mouse lung tissues. In summary, this study demonstrates that established viral infection specifically induces excessive lactic acid production in the host. As a key regulatory molecule, lactic acid directly impairs the antiviral efficacy of IFN-α by targeting and inhibiting IFN-α-mediated JAK-STAT signaling activation. This regulatory mechanism is valid across multiple cell types and murine in vivo models. Furthermore, the consistent elevation of LAC levels in clinical patients with COVID-19, influenza, and other infections indicates that lactic acid is not merely a metabolic byproduct of infection but a key factor contributing to viral immune escape and disease progression—further highlighting the generality and clinical significance of this mechanism. 3.4 Lactic Acid Triggers the Proinflammatory Effects of Interferon Prior studies, along with our own data, have established that IFN therapy significantly exacerbates inflammation following established viral infection (Fig. 1 ). Given that LAC levels are progressively elevated in cells, murine models, and clinical patients over the course of infection (Fig. 3 D-F), and that LAC is a key regulatory factor inhibiting the antiviral activity of IFN-α (Fig. 3 ; Figure S5), we hypothesized that LAC may contribute to disease progression by amplifying IFN-α-induced inflammatory responses—prompting targeted investigations. We first performed correlation analyses on clinical samples from influenza and COVID-19 patients. LAC levels in patients were significantly positively correlated with inflammatory markers (interleukin-6 [IL-6], high-sensitivity C-reactive protein [CRP]) and tissue damage markers (creatine kinase-MB [CK-MB], lactic acid dehydrogenase [LDH]) (Fig. 4 A). This clinical association between elevated LAC, inflammation, and tissue injury provided a foundation for mechanistic inquiry. To define the central role of LAC in inflammatory regulation, we further investigated the inflammatory response characteristics of virus-infected cells in the presence of LAC (Fig. 4 B). Results showed that compared to viral infection alone, LAC treatment significantly promoted the expression of key proinflammatory cytokines including IL-1β , IL-1α , IL-6 , and TNF-α (Fig. 4 C; Figure S6A-C)—a phenomenon conserved across different viral infections and multiple cell types. To validate the in vivo relevance of these in vitro findings, we established a murine model of viral infection combined with LAC treatment (Fig. 4 D). Results showed that compared to viral infection alone, LAC treatment significantly enhanced viral replication (Fig. 4 E; Figure S7A and S7B)—consistent with our previous conclusion that "LAC impairs IFN-α antiviral activity by targeting and inhibiting IFN-α-mediated JAK-STAT signaling" (Fig. 3 ). More critically, LAC treatment significantly exacerbated tissue inflammatory responses and pathological damage in mice compared to viral infection alone (Fig. 4 F and 4 G; Figure S7C-E). These in vivo experiments further confirm that LAC not only inhibits IFN-α antiviral effects but also amplifies its proinflammatory activity, accelerating disease progression. Since the NF-κB signaling pathway is the core mediator of proinflammatory cytokine production, we further evaluated the regulatory effect of IFN-α treatment on cellular inflammatory responses in the presence of LAC (Fig. 4 H). Strikingly, LAC significantly enhanced IFN-α-induced phosphorylation of p65—a key upstream molecule in the NF-κB signaling pathway (Fig. 4 I; Figure S8A-D)—and upregulated the expression of downstream IL-1β , IL-1α , IL-6 , and TNF-α . In contrast, treatment with LAC or IFN-α alone failed to significantly activate the NF-κB pathway or the expression of related proinflammatory cytokines (Fig. 4 I; Figure S8A-D). This reveals that IFN-α alone is insufficient to drive substantial inflammation, but in the presence of LAC, it acquires potent proinflammatory capacity, even capable of inducing a cytokine storm in the absence of infection. To further validate these findings, exclude confounding factors such as viruses, and confirm the direct regulatory role of LAC in IFN-α proinflammatory effects, we established a murine model of LAC combined with IFN-α treatment in the absence of viral infection (Fig. 4 J). Results showed that IFN-α monotherapy had no significant effect on mouse survival and did not induce obvious tissue damage or inflammatory responses (Fig. 4 K-M). In contrast, compared to the IFN-α monotherapy group, the LAC + IFN-α combination group exhibited significantly reduced mouse survival (Fig. 4 K), with severe inflammatory infiltration and pathological damage in liver tissues (Fig. 4 L and 4 M). This critical evidence further confirms that LAC can directly trigger IFN-α's proinflammatory effects, inducing cytokine storms and exacerbating tissue damage even without viral involvement. In summary, this study clearly demonstrates that LAC is a key regulatory factor underlying IFN-α therapeutic failure and adverse effects. It exacerbates virus-associated pathological damage and impairs clinical outcomes through a dual mechanism: ( 1 ) impairing IFN-α antiviral activity by inhibiting IFN-α-mediated JAK-STAT signaling, thereby promoting viral replication; ( 2 ) directly triggering IFN-α's proinflammatory potential to induce cytokine storms, exacerbating tissue inflammation and pathological damage, ultimately leading to host death—even in the absence of viral infection. This finding not only reveals the key mechanism underlying adverse effects of IFN-α in clinical therapy but also suggests that LAC levels may serve as a potential biomarker for evaluating IFN-α treatment safety. It provides important theoretical basis and clinical reference for optimizing antiviral therapeutic strategies and improving patient prognosis. 3.5 Lactic Acid Attenuates Antiviral JAK-STAT Signaling Through PKA-SIRT1 Axis Activation Having identified lactic acid as a key driver of impaired interferon-α (IFN-α) antiviral function and exacerbated tissue inflammatory damage, this study further dissected its underlying molecular mechanism. Given reports that LAC can modulate target protein function via direct lactylation modification, we first investigated whether it modulates JAK-STAT pathway activation by inducing lactylation of signal transducer and activator of transcription 1 (STAT1). A549 and PM cells were treated with gradient concentrations of LAC, and results showed no significant differences in STAT1 protein expression across groups (Fig. 5 A and 5 B), indicating that LAC does not affect STAT1 protein synthesis or stability. Furthermore, HA-tagged STAT1 recombinant plasmid was overexpressed in 293T cells, and co-immunoprecipitation (Co-IP) assays confirmed that STAT1 was not lactylated (Fig. 5 C). These findings clearly rule out the possibility that LAC regulates STAT1 function via direct lactylation modification. LAC has also been shown to exert indirect regulatory effects by activating multiple signaling pathways—for example, initiating the downstream GPR132-PKA pathway via G protein-coupled receptor 132 (GPR132)( 24 – 26 ), activating the PKA signaling pathway after transmembrane transport mediated by monocarboxylate transporter 1 (MCT1)( 27 ), or regulating gene transcription via the histone acetyltransferase p300 as a key substrate for histone lactylation( 28 ) (Figure S9A). However, which component of these signaling pathways mediates LAC's inhibitory effect on the IFN pathway remains unclear. To address this question, A549 and PM cells were pretreated with the GPR132 antagonist Telmisartan, PKA inhibitor H-89, or MCT1 inhibitor AZD3965, followed by co-stimulation with LAC and IFN-α. Results demonstrated that targeted inhibition of the GPR132-PKA pathway and MCT1 in A549 and PM cells significantly restored STAT1 phosphorylation levels and the expression of ISG15 (Fig. 5 D). This demonstrates that LAC's inhibition of the IFN pathway is dependent on MCT1-mediated transmembrane transport, GPR132 receptor binding, and downstream PKA signaling activation—with PKA downstream molecules potentially serving as key regulatory nodes. To identify the key downstream mediator of PKA, we performed screening of downstream factors. Previous studies have shown that downstream of PKA can regulate biological functions by activating AMPK( 29 ) and SIRT1( 30 ). Concomitantly, lactate can also directly induce histone lactylation via p300. We pretreated cells and lung with the AMPK inhibitor Compound C, SIRT1 inhibitor EX527, or p300 inhibitor C646, followed by co-stimulation with LAC and IFN-α. Results showed that only the SIRT1 inhibitor EX527 effectively restored IFN-α-mediated JAK-STAT pathway activation (Fig. 5 E; Figure S9B), while AMPK and p300 inhibitors had no such effect—confirming that SIRT1 is the key downstream molecule mediating LAC's inhibition of the IFN pathway. Meanwhile, high-throughput sequencing analysis of bronchoalveolar lavage fluid (BALF) from COVID-19 patients and healthy controls demonstrated a significant upregulation of SIRT1 in the patient cohort compared other SIRT families. (Figure S9C). Previous studies have demonstrated that SIRT1, an NAD+-dependent deacetylase, negatively regulates the JAK-STAT pathway by promoting STAT1 acetylation and inhibiting its phosphorylation( 31 ), but its role in LAC-mediated IFN inhibition has not been reported. We found that LAC treatment significantly upregulated SIRT1 protein expression in multiple cell lines including A549 and PM (Fig. 5 F). At the in vivo level, SIRT1 expression was also significantly increased in mouse liver and kidney tissues following intraperitoneal LAC injection (Fig. 5 G and 5 H). Consistent in vitro and in vivo results confirm that LAC specifically induces excessive SIRT1 expression. Lastly, mice were pretreated with the PKA inhibitor H-89 and the SIRT1 inhibitor EX527, followed by co-stimulation with LAC and IFN-α. Our results demonstrated that lactic acid significantly suppressed the activation of the JAK-STAT signaling pathway in mouse lung tissues, while H-89 and EX527 effectively reversed this lactic acid-mediated inhibitory effect. (Fig. 5 I). In summary, this study demonstrates that excessive LAC accumulation induced by viral infection can specifically upregulate SIRT1 expression via PKA activation, inhibiting STAT1 phosphorylation and blocking the JAK-STAT antiviral signaling pathway—ultimately impairing IFN-α antiviral efficacy and exacerbating tissue inflammatory damage (Fig. 5 J). This mechanism not only reveals a potential strategy for viral escape from host antiviral immunity but also provides a key pathophysiological explanation for the poor antiviral response and inflammation-related adverse effects (e.g., hepatorenal impairment) observed in clinical IFN-α therapy. Viral infection patients often exhibit abnormally elevated LAC metabolism in clinical settings, and the LAC-SIRT1-JAK-STAT regulatory axis identified in this study suggests that targeting lactic acid metabolism may serve as a potential intervention strategy to improve IFN-α therapeutic response and reduce treatment-related damage. 3.6 Combined Administration of Stiripentol and IFN-α Synergistically Inhibits Viral Replication and Inflammatory Responses Previous studies, including our own, have clearly identified LAC as a key regulatory factor that impairs IFN-α antiviral efficacy and exacerbates virus-induced tissue inflammatory damage. Based on this, targeted inhibition of lactic acid production may serve as an effective strategy to restore IFN-α antiviral activity and alleviate inflammation-related damage associated with viral replication. Building on these findings, we developed a specific combined therapeutic regimen of IFN-α plus the lactic acid dehydrogenase (LDH) inhibitor stiripentol. As an FDA-approved clinical drug for the treatment of severe myoclonic epilepsy in infants( 32 ), stiripentol’s classical mechanism of action involves regulating γ-aminobutyric acid (GABA)-ergic nervous system function to inhibit excessive neuronal excitation( 33 , 34 ). Critically, it also inhibits the property of inhibiting the activity of LDH—a key enzyme in lactic acid metabolism—thereby blocking lactic acid production at its source. Previous studies have confirmed that stiripentol can downregulate the lactylation modification level of NBS1 protein by inhibiting lactic acid production in gastric cancer cells, reduce tumor cell DNA repair capacity, and thereby overcome tumor radioresistance and chemoresistance( 35 ). Stiripentol was verified to effectively suppress lactic acid production induced by viral infection in cell and mice(Fig. 6 A). Subsequently, we treated it with stetamentanol as shown in the figure (Fig. 6 B). Results showed that compared to pre-infection IFN-α monotherapy, IFN-α alone almost lost its antiviral activity after infection was established. In contrast, stiripentol treatment effectively rescued IFN-α-mediated JAK-STAT signaling activation post-infection and significantly inhibited viral replication (Fig. 6 C)—confirming the core role of the combined therapy in reversing IFN-α antiviral failure. To evaluate the in vivo potential of the combined therapy, we established a murine viral infection model. After infection was established, mice were treated with IFN-α monotherapy, stiripentol monotherapy, or their combination (Fig. 6 D). Results confirmed that post-infection, IFN-α monotherapy failed to effectively activate the JAK-STAT signaling pathway or induce antiviral gene expression (Fig. 6 E, S10A-B). Stiripentol monotherapy partially restored IFN-α antiviral function and upregulated the expression of antiviral factors (Figure S10A and S10B). Strikingly, the IFN-α + stiripentol combination group exhibited the optimal effect, even when administered post- infection, it still drove IFN-α to efficiently activate the JAK-STAT antiviral signaling pathway, significantly induce antiviral factor expression, and inhibit viral replication (Fig. 6 E; Figure S10A and S10B)—fully validating the in vivo antiviral efficacy of the combined therapy. Given previous evidence that LAC can directly trigger IFN-α’s proinflammatory effects, inducing cytokine storms and exacerbating tissue damage even in the absence of virus, we further evaluated the regulatory role of the combined therapy on post-infection inflammatory responses. Results showed that compared to the high levels of inflammatory cytokines in the IFN-α monotherapy group, stiripentol monotherapy partially downregulated inflammatory cytokine expression. In contrast, the combination therapy group significantly suppressed infection-induced cytokine storms (Figure S11A and S11B), demonstrating potent anti-inflammatory activity. Histopathological analysis further revealed that compared to viral infection alone, IFN-α monotherapy post-infection significantly increased inflammatory neutrophil infiltration in mouse liver and lung tissues, inducing more severe histopathological damage (Fig. 6 F and 6 G; Figure S11C-E). Stiripentol monotherapy had no significant improvement on tissue damage or inflammatory neutrophil infiltration. However, compared to the infection-only group and each monotherapy group, the combination therapy group showed significantly reduced inflammatory neutrophil infiltration and markedly alleviated histopathological damage in liver and lung tissues (Fig. 6 F and 6 G; Figure S11C-E)—achieving synergistic benefits of "antiviral activity + inflammation control". A core bottleneck in current clinical application of IFN-α lies in the impracticality of prophylactic intervention strategies widely used in preclinical studies (e.g., "pre-infection pretreatment" or "concomitant administration"). Prophylactic administration prior to infection cannot be routinely performed clinically, nor can the optimal timing of administration be accurately determined. Clinical therapeutic interventions for confirmed patients mostly initiate after infection is established, where IFN-α monotherapy often leads to a dualseed dilemma of "antiviral failure + enhanced inflammatory side effects". The core advantage of this combined therapy is its significant efficacy even when intervention is initiated post-infection—completely breaking the strict restriction of traditional IFN-α therapy on administration timing. It effectively reverses LAC-mediated IFN-α antiviral inhibition and proinflammatory adverse effects, overcomes some core limitations of clinical IFN-α application, and significantly improves late-stage infection treatment efficacy while reducing the side effects of monotherapy. This not only provides a novel strategy for the development of broad-spectrum antiviral drugs but also offers new therapeutic options and directions for addressing current and future viral pandemics—presenting a practical solution to the long-standing problem of limited clinical application of IFN-α in antiviral therapy. Discussion The global spread of emerging and re-emerging viral pandemics continues to pose a severe threat to public health security. While vaccines and traditional antiviral drugs have provided partial solutions for combating known viruses, their inherent limitations have become increasingly prominent: vaccines require a long development cycle, are vulnerable to viral mutation and escape, and can only target specific viral subtypes; traditional antiviral drugs mostly target specific viral proteins, facing challenges such as rapid development of drug resistance and ineffectiveness against unknown viruses. These shortcomings collectively highlight the urgency and strategic significance of developing broad-spectrum antiviral agents that are relatively independent of viral types. Since the first discovery of interferons (IFNs) by Isaacs and Lindenmann in 1957( 36 ), their pivotal role as core effector molecules of the host innate antiviral immunity has been fully validated through decades of research, making them a central target for studies on antiviral immune mechanisms and clinical translation. In contrast to their prominent role in preclinical research, the current clinical application of IFNs is extremely limited. They are only approved for a few diseases such as chronic hepatitis B and chronic hepatitis C, and are rarely the first-line treatment of choice. For acute viral infections with high incidence and wide impact, such as acute influenza and COVID-19, IFNs are not recommended for routine use; instead, they are strictly restricted due to limited efficacy and high risk of adverse reactions. To date, the core mechanisms underlying IFN clinical failure and side effects remain unclear, and there is a lack of effective solutions to address this clinical dilemma, which has greatly hindered the full realization of their clinical value and the development of broad-spectrum antiviral strategies. Through systematic validation using clinical samples, multi-viral infection cell models, and murine models, this study found that the prophylactic intervention modes such as "pre-infection pretreatment" or "concomitant administration" widely used in preclinical research are rarely feasible in clinical practice. Prophylactic use before infection is difficult to implement, and the optimal timing of administration is even more challenging to determine. Clinical therapeutic interventions for confirmed patients mostly occur at the established infection stage, where drug administration results in a dual negative effect of "antiviral failure + enhanced side effects"—this is the core reason why the basic efficacy of IFNs cannot be translated into clinical outcomes. Mechanistically, this study identified host-derived lactic acid induced by viral infection as the key driver of the clinical bottleneck of IFN therapy, and clarified its dual regulatory mechanism of "LAC-SIRT1-JAK-STAT/LAC-NF-κB". Ultimately, a transformative combination therapy of IFN plus stiripentol was developed, providing a novel strategy for broad-spectrum antiviral treatment. This study confirmed that IFN therapeutic efficacy is highly dependent on the timing of infection, which provides a key entry point for explaining the discrepancy between its preclinical and clinical efficacy. In vitro experiments demonstrated that IFN pretreatment can effectively inhibit the replication of various viruses and reduce inflammatory responses in multiple cell types; however, administration after the establishment of viral infection not only significantly impairs antiviral activity but also exacerbates cytokine storms and tissue damage. In vivo experiments further verified the universality of this rule, suggesting that clinical therapeutic interventions for confirmed patients after established infection are the core cause of poor IFN efficacy. In-depth studies revealed that virus-induced LAC at concentrations far exceeding the normal physiological level serves as the central factor mediating the abnormal function of IFN. Our experimental model was established to simulate clinical infection scenarios. Particularly, with respect to the lactate stimulation intervention step, we designed the treatment sequence based on the clinical pathological process in which lactate is first induced upon infection prior to interferon administration. Subsequent experimental validation confirmed that interferon alone failed to trigger an inflammatory response; instead, lactate produced during viral infection acts as the critical synergistic factor mediating the induction of inflammatory reactions. Transcriptome analysis and metabolic detection confirmed that different viral infections universally activate the host glycolytic pathway, leading to a significant increase in LAC levels in cells and serum—this phenomenon was validated in clinical samples from COVID-19 and influenza patients, highlighting its pathological relevance. This further indicates that LAC, previously considered a mere metabolic byproduct, may play a key role in infection-related disease progression. Mechanistic experiments clarified that LAC impairs IFN therapeutic effects through a dual mechanism: ( 1 ) LAC induces excessive SIRT1 expression via the GPR132-PKA signaling pathway, thereby inhibiting STAT1 phosphorylation, blocking the activation of the JAK-STAT antiviral signaling pathway, and directly impairing the viral inhibitory capacity of IFNs; ( 2 ) LAC synergizes with IFNs to efficiently activate the NF-κB pathway, inducing storms of proinflammatory cytokines such as IL-1β and IL-6, and exacerbating tissue damage. Notably, combined treatment with LAC and IFNs can induce inflammatory damage and mouse death even in the absence of viral infection, confirming that this proinflammatory effect is independent of viral replication and serves as a key inducer of IFN clinical side effects. This mechanism not only clarifies the critical role of LAC in regulating IFN antiviral function but also reveals the central role of SIRT1 in LAC-mediated IFN signaling inhibition, providing a clear molecular target for targeted intervention. Based on the above mechanisms, this study innovatively developed a "IFN combined with lactic acid dehydrogenase (LDH) inhibitor stiripentol" combination therapy, realizing a complete chain of "mechanism-target-drug". As an FDA-approved clinical drug, stiripentol's property of inhibiting LDH activity and blocking LAC production was first applied in antiviral therapy. In vitro and in vivo experiments confirmed that even when intervention is initiated after the establishment of viral infection, this combination therapy can effectively reduce LAC levels, restore IFN-mediated JAK-STAT pathway activation, and significantly inhibit viral replication; meanwhile, it can potently block LAC-IFN synergistically induced cytokine storms, alleviate hepatopulmonary pathological damage, and achieve dual benefits of "efficient antiviral activity + precise inflammation control". The core advantage of this regimen lies in breaking the strict restriction of IFN on administration timing, solving the practical clinical problem of "difficulty in early intervention". Additionally, the clinical application basis of stiripentol reduces translational risks, enabling the rapid advancement of clinical research. The aforementioned combination therapy provides novel targets and technical strategies for the development of broad-spectrum antiviral agents. Beyond its substantial antiviral activity, IFN also exerts potent effects in contexts such as antitumor therapy and antibacterial infection. Previous studies have demonstrated that lactic acid is similarly highly expressed in the tumor microenvironment and infected lesions( 37 , 38 ), indicating that lactic acid may also interact with interferons. Based on this observation, the relevant mechanism of action and combined therapeutic regimen proposed herein are also expected to open new avenues and provide innovative strategies for the treatment of tumors and other diseases. This offers novel therapeutic approaches and drug options for addressing existing and future unknown diseases as well as viral pandemics, holding significant clinical translational value and public health implications. Materials and methods Comprehensive information regarding the reagents, consumables, antibodies, and other relevant materials utilized in the experiments is available in Table S1 . Patient samples All clinical samples were collected from the Shiyan People's Hospital, Hubei Medical University. Lactate levels, inflammatory factor levels, and myocardial injury markers were collected 210 COVID-19 patients, 23 influenza patients (on Day 1 and Day 3 of illness respectively), and 58 healthy controls from Shiyan People's Hospital, who were admitted between January 2022 and December 2023. All samples were collected with the patients' informed consent and signed written consent forms. The experiment was approved by the Ethics Committee of Shiyan People's Hospital Affiliated to Hubei University of Medicine, and complied with the ethical standards established in the Declaration of Helsinki. Cells In this study, various cell lines, including HEK293T, VERO, A549 and MDCK were obtained from the American Type Culture Collection (ATCC). Primary peritoneal macrophages were isolated from mice 5 days following intraperitoneal injection of thioglycollate broth medium. Lung fibroblasts were isolated by mincing mouse lungs and digesting with Hank's solution. These were cultured in DMEM media. All media were supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2. It’s noteworthy that all cell lines used in the study were regularly tested and confirmed to be free from mycoplasma contamination. Viruses Influenza H1N1-PR stocks were propagated in Serum Free Medium for MDCK Cells. VSV-GFP propagated in Serum Free Medium for VERO Cells. Animals All mice were maintained in a specific-pathogen-free facility with a 12-hour light/dark cycle and were allowed ad libitum access to food and water. Six-week-old male C57BL/6J wild-type mice were purchased from Changsheng Biotechnology (Liaoning, China). Mice were infected with H1N1 virus via intranasal instillation and with VSV virus via intraperitoneal injection. Additionally, mice were administered IFN-α, LAC and stiripentol via intraperitoneal injection for stimulation. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Hubei University of Medicine. Infection and transfection For infection, A549, PM and MLF cells were infected with DMEM containing viruses with a multiplicity of infection (MOI). For transfection, plasmids were transfected by using Lipofectamine 3000 according to the manufacturers instructions, cells were harvested or further treatment as indicated. Immunoblotting (IB) Cells were harvested and lysed with lysis buffer (150 mM NaCl, 50 mM Tris-HCl [PH 7.4], 1% Triton X-100, 1mM EDTA [pH 8.0], 0.1% SDS supplemented with a protease inhibitor cocktail) for 30 min at 4°C. The supernatants were collected by centrifugation at 12000 g for 25 min at 4°C. Protein concentrations were determined using the Bradford method. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% non-fat milk dissolved in TBST (phosphate-buffered saline with 0.1% Tween 20), it was incubated with the primary Abs, followed by horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG. The proteins were detected on a BIO-RAD ChemiDoc Imaging System by using Enhanced chemiluminescence (ECL). Immunoprecipitation(IP) For protein-protein interactions assays, cells were lysed in RIPA lysis buffer (150 mM NaCl, 0.5% NP-40, 5 mM EDTA) containing a mixture of protease inhibitor (Roche). Primary antibodies were incubated with Protein A/G agarose beads for 30 min at room temperature, followed by incubation with cell lysates for 3 hours with rotation at room temperature (RT). The beads were washed four times with lysis buffer and analyzed by IB. Immunohistochemistry(IHC) analysis Thin sections of hepatic and pulmonary tissues were treated to remove paraffin and restore hydration. They were then placed in a sodium citrate buffer with a pH of 6.0 for antigen retrieval and allowed to cool. Following this, endogenous peroxidase activity was suppressed for a duration of 10 min through the application of a 0.3% (v/v) hydrogen peroxide solution in methanol. After being incubated with a 10% goat serum blocking solution for 30 min, the samples were subjected to Ly-6G (1:200) antibodies overnight. Following two rounds of washing, the samples underwent a 30 min incubation at room temperature with a secondary antibody diluted at a ratio of 1:100. The cell nuclei were subsequently stained with hematoxylin following the color development using the DAB kit. RNA extraction and quantitative RT-PCR Total RNA was extracted from cells or tissues that have been processed with Trizol. 0.6 µg of total RNA was reversed transcribed to synthesize cDNA by HiScipt Ⅱ Q RT SuperMix, using ChamQ SYBR qPCR Master Mix for qPCR. Real-time quantitative PCR was run in duplicate using iTaq SYBRGreen (Bio-Rad), following manufacturer’s instructions. Data are presented as relative mRNA abundance normalized to GAPDH expression in each sample.( Primer sequence information is shown in Table S2) RNA-sequencing and data processing Virus-infected cells were immediately transferred into 1 ml Trizol, snap-frozen in liquid nitrogen, and kept at − 80°C. RNA extraction was performed by GeneDenovo (QIAGEN, China). RNA integrity number (RIN) was determined using the Agilent RNA 6000 Nano Kit for quality control and all samples were above 8.5. After sequencing, samples were aligned to the Ensembl_release111 Genome. For the pathway analysis, GSEA (4.2.3) was used to identify pathway enrichment among the total genes expressed in the PM cells infected with VSVG or mock. Lactate measurements L-lactic acid levels in cells and mouse tissues were measured using the L-lactic acid assay kit according to the manufacturer’s instructions. Enzyme-linked immunosorbent assay Purified proteins were immobilized onto 96-well plates in a carbonate buffer (200 ng per well) and incubated at 4°C overnight. After five times washing with PBST (PBS with 0.05% Tween 20), blocking was performed using 1% BSA in PBST. Subsequently, Mouse serum samples were added and incubated at room temperature for 2 h. Following five times washing, Secondary antibodies were added for 1 h. Then, the HRP diluted in PBST with 1% BSA was added and incubated for an additional hour. After five times washing, the TMB substrate was applied, and the reaction was stopped by adding 1 M H2SO4. Absorbance at 450 nm was measured and recorded. The obtained data were analyzed using GraphPad Prism 9. H&E staining of liver and lung tissues Hepatic and pulmonary tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4-µm slices. Sections were deparaffinized in xylene, hydrated through a graded ethanol series, stained with filtered Harris hematoxylin for 8 min and 0.5% eosin Y for 3 min, followed by dehydration and clearing. Stained sections were mounted with neutral balsam and visualized under a light microscope. Statistical analysis The data are presented as the means ± standard deviations from at least three independent experiments. Comparisons of two groups were performed by using two-tailed unpaired Student’s t test. Comparisons of multiple groups were performed by using one-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test unless otherwise indicated. Statistical analysis was performed using the GraphPad Prism 7 software package. For all analyses, a p-value < 0.05 was considered to indicate statistical significance Declarations Author contributions K.L., and Z.L. contributed to the study concept and design. M.T., S.C. and J.H. carried out most of the experiments. X.W., M.L., X.L., Z.C., X.X., S.L, M.L., C.H., N.W., Z.Z., and W.D. analysed the data with help from K.L. and Z.L. contributed to the drafting of the manuscript. Q.T., contributed to the reagents. W.D., J.W., and Z.L. contributed to edit the manuscript. All authors contributed to the article and approved the submitted version. Acknowledgements This work was supported by research grants from National Natural Science Foundation of China (32188101, 32400131), Hubei Provincial Natural Science Foundation (2023BCB058, JCZRLH202600075), Natural Science Foundation of Hubei Provincial Department of Education (D20242105), China Postdoctoral Science Foundation (2024T170687, 2024M752482, GZB20230541) and the Open Research Fund of State Key Laboratory of Virology and Biosafety (SKLVKF2025014). Competing interests The authors declare that they have no competing interests. Data Availability Statement The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors. Ethics approval All experiments involving mice were performed according to the institutional animal ethics committee protocol. References Del Rio C, Collins L F, Malani P. Long-term health consequences of covid-19 [J]. Jama, 2020, 324(17): 1723-1724. Al-Aly Z, Davis H, McCorkell L, et al. Long covid science, research and policy [J]. Nat Med, 2024, 30(8): 2148-2164. Pestka S. The interferons: 50 years after their discovery, there is much more to learn [J]. J Biol Chem, 2007, 282(28): 20047-20051. McNab F, Mayer-Barber K, Sher A, et al. Type i interferons in infectious disease [J]. Nat Rev Immunol, 2015, 15(2): 87-103. Li F, Wang Q J, Zhu B L, et al. Antiviral effects of rhifn-alpha 1 against seven influenza viruses [J]. Zhongguo Yao Li Xue Bao, 1999, 20(8): 709-714. Müller U, Steinhoff U, Reis L F, et al. Functional role of type i and type ii interferons in antiviral defense [J]. Science, 1994, 264(5167): 1918-1921. Pan H, Peto R, Henao-Restrepo A M, et al. Repurposed antiviral drugs for covid-19 - interim who solidarity trial results [J]. N Engl J Med, 2021, 384(6): 497-511. Asselah T, Lada O, Moucari R, et al. Interferon therapy for chronic hepatitis b [J]. Clin Liver Dis, 2007, 11(4): 839-849, viii. Shapiro S, Gershtein V, Elias N, et al. Mrna cytokine profile in peripheral blood cells from chronic hepatitis c virus (hcv)-infected patients: Effects of interferon-alpha (ifn-alpha) treatment [J]. Clin Exp Immunol, 1998, 114(1): 55-60. Waldmann T A. Cytokines in cancer immunotherapy [J]. Cold Spring Harb Perspect Biol, 2018, 10(12). Locke M C, Fox L E, Dunlap B F, et al. Interferon alpha, but not interferon beta, acts early to control chronic chikungunya virus pathogenesis [J]. J Virol, 2022, 96(1): e0114321. Cooke G S, Hung L M, Flower B, et al. Treatment options to support the elimination of hepatitis c: An open-label, factorial, randomised controlled non-inferiority trial [J]. Lancet, 2025, 405(10491): 1769-1780. Finter N B, Chapman S, Dowd P, et al. The use of interferon-alpha in virus infections [J]. Drugs, 1991, 42(5): 749-765. Tannock G A, Gillett S M, Gillett R S, et al. A study of intranasally administered interferon a (rifn-alpha 2a) for the seasonal prophylaxis of natural viral infections of the upper respiratory tract in healthy volunteers [J]. Epidemiol Infect, 1988, 101(3): 611-621. Galbraith M D, Kinning K T, Sullivan K D, et al. Specialized interferon action in covid-19 [J]. Proc Natl Acad Sci U S A, 2022, 119(11). Hayden F G, Albrecht J K, Kaiser D L, et al. Prevention of natural colds by contact prophylaxis with intranasal alpha 2-interferon [J]. N Engl J Med, 1986, 314(2): 71-75. Davidson S, McCabe T M, Crotta S, et al. Ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment [J]. EMBO Mol Med, 2016, 8(9): 1099-1112. Kalil A C, Mehta A K, Patterson T F, et al. Efficacy of interferon beta-1a plus remdesivir compared with remdesivir alone in hospitalised adults with covid-19: A double-bind, randomised, placebo-controlled, phase 3 trial [J]. Lancet Respir Med, 2021, 9(12): 1365-1376. Wong L R, Perlman S. Immune dysregulation and immunopathology induced by sars-cov-2 and related coronaviruses - are we our own worst enemy? [J]. Nat Rev Immunol, 2022, 22(1): 47-56. Randall R E, Goodbourn S. Interferons and viruses: An interplay between induction, signalling, antiviral responses and virus countermeasures [J]. J Gen Virol, 2008, 89(Pt 1): 1-47. Kim Y M, Shin E C. Type i and iii interferon responses in sars-cov-2 infection [J]. Exp Mol Med, 2021, 53(5): 750-760. Xiao H, Killip M J, Staeheli P, et al. The human interferon-induced mxa protein inhibits early stages of influenza a virus infection by retaining the incoming viral genome in the cytoplasm [J]. J Virol, 2013, 87(23): 13053-13058. Kristiansen H, Scherer C A, McVean M, et al. Extracellular 2'-5' oligoadenylate synthetase stimulates rnase l-independent antiviral activity: A novel mechanism of virus-induced innate immunity [J]. J Virol, 2010, 84(22): 11898-11904. Liu W, Zhang S, Li Q, et al. Lactate modulates iron metabolism by binding soluble adenylyl cyclase [J]. Cell Metab, 2023, 35(9): 1597-1612.e1596. Ngai D, Schilperoort M, Tabas I. Efferocytosis-induced lactate enables the proliferation of pro-resolving macrophages to mediate tissue repair [J]. Nat Metab, 2023, 5(12): 2206-2219. Cantó C, Gerhart-Hines Z, Feige J N, et al. Ampk regulates energy expenditure by modulating nad+ metabolism and sirt1 activity [J]. Nature, 2009, 458(7241): 1056-1060. Hahn E L, Halestrap A P, Gamelli R L. Expression of the lactate transporter mct1 in macrophages [J]. Shock, 2000, 13(4): 253-260. Zhang D, Tang Z, Huang H, et al. Metabolic regulation of gene expression by histone lactylation [J]. Nature, 2019, 574(7779): 575-580. Medina E A, Oberheu K, Polusani S R, et al. Pka/ampk signaling in relation to adiponectin's antiproliferative effect on multiple myeloma cells [J]. Leukemia, 2014, 28(10): 2080-2089. Gerhart-Hines Z, Dominy J E, Jr., Blättler S M, et al. The camp/pka pathway rapidly activates sirt1 to promote fatty acid oxidation independently of changes in nad(+) [J]. Mol Cell, 2011, 44(6): 851-863. Yu S S, Tang R C, Zhang A, et al. Deacetylase sirtuin 1 mitigates type i ifn- and type ii ifn-induced signaling and antiviral immunity [J]. J Virol, 2024, 98(3): e0008824. Chiron C, Marchand M C, Tran A, et al. Stiripentol in severe myoclonic epilepsy in infancy: A randomised placebo-controlled syndrome-dedicated trial. Sticlo study group [J]. Lancet, 2000, 356(9242): 1638-1642. Fisher J L. The effects of stiripentol on gaba(a) receptors [J]. Epilepsia, 2011, 52 Suppl 2(0 2): 76-78. Fisher J L. The anti-convulsant stiripentol acts directly on the gaba(a) receptor as a positive allosteric modulator [J]. Neuropharmacology, 2009, 56(1): 190-197. Chen H, Li Y, Li H, et al. Nbs1 lactylation is required for efficient DNA repair and chemotherapy resistance [J]. Nature, 2024, 631(8021): 663-669. Isaacs A, Lindenmann J. Virus interference. I. The interferon [J]. Proc R Soc Lond B Biol Sci, 1957, 147(927): 258-267. Li J, Li Z, Zhang X, et al. Histone lactylation bridges metabolic reprogramming with chromatin-immune crosstalk in triple-negative breast cancer [J]. Cancer Lett, 2025, 639: 218227. Peng X, He Z, Yuan D, et al. Lactic acid: The culprit behind the immunosuppressive microenvironment in hepatocellular carcinoma [J]. Biochim Biophys Acta Rev Cancer, 2024, 1879(5): 189164. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMaterialsfinal1.docx Host-Derived Lactic Acid Disrupts IFN Clinical Efficacy via Antiviral Inhibition and Proinflammatory Amplification GraphAbstract.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weixing","middleName":"","lastName":"Du","suffix":""},{"id":608532575,"identity":"e5b73bec-72c4-4015-971a-cf9a1da45409","order_by":14,"name":"Ziyi Zhang","email":"","orcid":"","institution":"Hubei University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ziyi","middleName":"","lastName":"Zhang","suffix":""},{"id":608532576,"identity":"c6589eb0-9d0d-443a-8078-592eaf334101","order_by":15,"name":"Kailang Wu","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Kailang","middleName":"","lastName":"Wu","suffix":""},{"id":608532577,"identity":"4f748365-c861-4a36-bd92-1cb9bfce3594","order_by":16,"name":"Ke Lan","email":"","orcid":"https://orcid.org/0000-0002-0384-8598","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Lan","suffix":""}],"badges":[],"createdAt":"2026-02-18 03:00:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8905265/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8905265/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105055503,"identity":"86f419f7-8cd4-4763-ab3b-dfff7d193fd2","added_by":"auto","created_at":"2026-03-20 11:34:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10314912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFN-α Fails to Protect Against Established Viral Infection and Promotes Inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of IFN-α treatment timing in virus-infected cell models. A549 cells, peritoneal macrophages (PMs), and murine lung fibroblasts (MLFs) were infected with VSV or H1N1, followed by treated with 2 nM IFN-α at 2 h prior to infection, 4 h post-infection, or 12 h post-infection.\u003c/p\u003e\n\u003cp\u003e(B and C) Western blot analysis assessing the effect of IFN-α (2 nM) administration at the indicated time points (–2 h, +4 h, +12 h relative to infection) on the replication of VSV and H1N1 in A549 cells and PMs.\u003c/p\u003e\n\u003cp\u003e(D) ELISA quantification of IL-1β levels in A549 cells treated with IFN-α (2 nM) at indicated time points (-2 h, +24 h, +48 h). Data are presented as mean ± standard deviation (mean ± SD); n = 3. ns = no significant, ∗∗∗p \u0026lt; 0.001, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e(E) Schematic of IFN-α treatment at different time points in virus-infected murine models. C57BL/6 mice were infected with VSV or H1N1, and administered IFN-α (200 ng per mouse) at 2 h before infection, 24 h post-infection, or 48 h after infection.\u003c/p\u003e\n\u003cp\u003e(F) Western blot analysis and densitometric quantification of H1N1 viral replication in lung tissues of mice treated with IFN-α (200 ng per mouse) at the indicated time points (–2 h, +24 h, +48 h). Data are presented as mean ± SD; n = 3. ∗p \u0026lt; 0.05, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e(G) Histopathological analysis of lung tissue (Hematoxylin and Eosin [H\u0026amp;E] staining) from mice infected with H1N1 and treated with IFN-α (200 ng per mouse) at different time points (-2 h, +24 h, +48 h).\u003c/p\u003e\n\u003cp\u003e(H) Analysis of inflammatory response in lung tissue (immunohistochemistry [IHC] for LY6G) from mice infected with H1N1 and treated with IFN-α (200 ng per mouse) at the indicated time points (–2 h, +24 h, +48 h). Quantification of LY6G-positive cells. Data are presented as mean ± SD; n = 3. ns = no significant, ∗∗p \u0026lt;0.01, ∗∗∗p \u0026lt; 0.001, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8905265/v1/db5f71c4029122b52ddce5eb.png"},{"id":105055502,"identity":"7d2e19ca-89e6-4399-9a63-767bd5cbb639","added_by":"auto","created_at":"2026-03-20 11:34:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2507264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablished Viral Infection Suppresses IFN-α Signaling Transduction​\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of IFN-α treatment timing in virus-infected cells. A549 cells were infected with VSV or H1N1, followed by treatment with 2 nM IFN-α at 12 h or 24 h post-infection.\u003c/p\u003e\n\u003cp\u003e(B) Western blot analysis of the expression of \u003cem\u003ep-STAT1\u003c/em\u003e and\u003cem\u003e ISG15\u003c/em\u003e in IFN-α-treated A549 cells with or without prior viral infection.​\u003c/p\u003e\n\u003cp\u003e(C) Schematic of IFN-α treatment in primary macrophages with or without infection. Peritoneal macrophages (PMs) were infected with VSV or H1N1, followed by treatment with 2 nM IFN-α at 12 h or 24 h post-infection. Uninfected PM cells were treated with 2 nM IFN-α and harvested at 0.5 h, 1 h, or 3 h post-treatment.​\u003c/p\u003e\n\u003cp\u003e(D) Western blot analysis of \u003cem\u003ep-STAT1\u003c/em\u003e expression in IFN-α-treated PMs under the conditions described in (C).​\u003c/p\u003e\n\u003cp\u003e(E) Schematic of IFN-α treatment in virus-infected mice. Mice were challenged with VSV or H1N1, followed by IFN-α treatment at 24 h post-challenge. Lung tissues were collected at 36 h post-challenge for analysis.​\u003c/p\u003e\n\u003cp\u003e(F) Quantitative real-time PCR (qRT-PCR) analysis of downstream factors of the IFN-α-mediated JAK-STAT signaling pathway in lung tissues from the mice described in (E). Data are presented as mean ± SD; n = 3. ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8905265/v1/403f15d7adf0ce822dca5903.png"},{"id":105055510,"identity":"22465152-7eae-4086-98e4-40aec9c9f02a","added_by":"auto","created_at":"2026-03-20 11:34:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3682501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactic Acid Mediates the Suppression of IFN-α Antiviral Signaling After Viral Infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Gene Set Enrichment Analysis (GSEA) depicting the top 20 enriched pathways in VSVG-infected PM cells versus mock. (n = 3 biological replicates).\u003c/p\u003e\n\u003cp\u003e(B) GSEA enrichment plot for the Hallmark Glycolysis pathway in VSVG-infected PM cells(n = 3 biological replicates).\u003c/p\u003e\n\u003cp\u003e(C) Heatmap showing the expression of glycolysis-related genes in VSVG-infected PM cells. (n = 3 biological replicates).\u003c/p\u003e\n\u003cp\u003e(D) Schematic diagram illustrating the effects of glycolysis inhibitor 2-DG, lactate dehydrogenase inhibitor Oxamate, and pyruvate transport inhibitor UK-5099 on the glycolysis pathway.\u003c/p\u003e\n\u003cp\u003e(E) Western blot analysis of key JAK-STAT signaling pathway components in A549 cells and PMs treated with IFN-α, with or without prior inhibition of glycolysis by 2-DG.\u003c/p\u003e\n\u003cp\u003e(F) RT-PCR analysis of key downstream antiviral factors (MX1, OAS1) of the JAK-STAT signaling pathway in PM cells infected with VSV and subsequently treated with IFN-α, in the presence of the glycolysis inhibitor 2-DG, lactate dehydrogenase inhibitor Oxamate, and pyruvate transport inhibitor UK-5099. Data are presented as mean ± SD; n = 3. ns = no significant, ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e(G) Intracellular LAC levels in A549 cells and MLFs following established infection with VSV or H1N1. Data are presented as mean ± SD; n = 3. ∗∗∗p \u0026lt; 0.001, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e(H) Serum LAC levels in mice following established infection with VSV or H1N1. Data are presented as mean ± SD; n = 3. ∗∗∗p \u0026lt; 0.001, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e(I) Analysis of blood LAC levels in clinical cohorts. (Left) LAC levels in COVID-19 patients versus healthy controls. (Right) Longitudinal measurement of LAC levels in influenza patients at hospitalization (Day 0) and three days post-diagnosis (Day 3). Data are presented as mean ± SD; n = 3. ns = no significant, ∗∗p \u0026lt; 0.01, ∗∗∗∗p\u0026lt; 0.0001.​\u003c/p\u003e\n\u003cp\u003e(J) Western blot analysis of key upstream and downstream factors of the JAK-STAT signaling pathway in cells treated with LAC and IFN-α.\u003c/p\u003e\n\u003cp\u003e(K) Schematic of \u003cem\u003ein vivo\u003c/em\u003e treatment with LAC and IFN-α. Mice were administered LAC, followed by IFN-α treatment and a second LAC dose.\u003c/p\u003e\n\u003cp\u003e(L) Western blot analysis and densitometric quantification of key upstream factors of the JAK-STAT signaling pathway in mice treated with LAC and IFN-α. Data are presented as mean ± SD; n = 3. ∗∗p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8905265/v1/23e0f3a7350d8a20b71ce3a3.png"},{"id":105055505,"identity":"060c56f6-438b-4485-b3f9-f378191b2430","added_by":"auto","created_at":"2026-03-20 11:34:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9399844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactic Acid Exacerbates Virus-Induced Inflammatory Responses​\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Correlation analysis of LAC, LDH, CRP, WBC, CK-MB, and IL-6 levels in COVID-19 and influenza patients. Data are presented as mean ± SD; n = 91. ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e(B) Schematic diagram of virus-infected cells treated with LAC concurrently. PMs were infected with VSV or H1N1 and treated with LAC simultaneously.\u003c/p\u003e\n\u003cp\u003e(C) Western blot analysis of \u003cem\u003eIL-1β\u003c/em\u003e expression in virus-infected cells treated with LAC as in (B).\u003c/p\u003e\n\u003cp\u003e(D) Schematic of virus-infected mice treated with LAC concurrently. Mice were infected with VSV or H1N1 and intraperitoneally injected with LAC for stimulation. Tail-tip blood was collected at 12 h post-infection, and mice were euthanized at 24 h post-infection to collect lung tissues and orbital blood.\u003c/p\u003e\n\u003cp\u003e(E-G) Western blot analysis, densitometric quantification of H1N1, H\u0026amp;E and IHC (LY6G) staining of lung tissues from virus-infected mice treated with LAC as in (D). Data are presented as mean ± SD; n = 3. ∗∗p \u0026lt; 0.01. Quantification of LY6G-positive cells. Data are presented as mean ± SD; n = 3. ∗∗p \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003e(H) Schematic of cells treated with IFN-α following LAC stimulation. PMS were stimulated with LAC for 24 h, followed by IFN-α treatment. Cells were harvested for subsequent analyses.\u003c/p\u003e\n\u003cp\u003e(I) Western blot analysis of \u003cem\u003ep65\u003c/em\u003e and phosphorylated \u003cem\u003ep65\u003c/em\u003e (\u003cem\u003ep-p65\u003c/em\u003e) in cells treated with IFN-α following LAC stimulation.\u003c/p\u003e\n\u003cp\u003e(J) Schematic of mice co-stimulated with LAC and IFN-α. Mice were stimulated with LAC at 0 h and 8 h, treated with IFN-α at 2 h, and euthanized at 12 h to collect liver tissues and orbital blood for detection and analysis.\u003c/p\u003e\n\u003cp\u003e(K) Survival curve analysis of mice co-stimulated with LAC and IFN-α. n = 9. ∗∗∗p \u0026lt; 0.001, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e(L and M) H\u0026amp;E and IHC (LY6G) staining of liver tissues from mice co-stimulated with LAC and IFN-α. Quantification of LY6G-positive cells. Data are presented as mean ± SD; n = 3. ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8905265/v1/d070cd0d3260d17fdb44bccd.png"},{"id":105055508,"identity":"3508b78b-d43b-4899-a96a-18c4bbc799f2","added_by":"auto","created_at":"2026-03-20 11:34:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4764273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactic Acid Attenuates Antiviral JAK-STAT Signaling Through PKA-SIRT1 Axis Activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A and B) Western blot analysis of STAT1 in cells (A549, PMs) treated with the indicated concentrations of LAC.\u003c/p\u003e\n\u003cp\u003e(C) Co-immunoprecipitation (Co-IP) analysis of STAT1 lactylation modification. Panlac: Rabbit Monoclonal anti-L-Lactyl Lysine, used for detecting lysine L-lactylation modification.\u003c/p\u003e\n\u003cp\u003e(D) Western blot analysis of \u003cem\u003eSTAT1\u003c/em\u003e phosphorylation (\u003cem\u003ep-STAT1\u003c/em\u003e) and \u003cem\u003eISG15\u003c/em\u003e expression in cells (A549, PMs) co-treated with GPR132-PKA pathway inhibitors (H-89, Telmisartan), MCT1 inhibitor (AZD3965), LAC, and IFN-α.\u003c/p\u003e\n\u003cp\u003e(E) Western blot analysis of STAT1 phosphorylation (\u003cem\u003ep-STAT1\u003c/em\u003e) and \u003cem\u003eISG15\u003c/em\u003e expression in cells (A549, PM) co-treated with GPR132-PKA-SIRT1 pathway inhibitors (Compound C, EX527), p300 inhibitor (C646), LAC, and IFN-α.\u003c/p\u003e\n\u003cp\u003e(F) Western blot and RT-PCR analyses of \u003cem\u003eSIRT1\u003c/em\u003e in cells (A549, PM) stimulated with LAC. Data are presented as mean ± SD; n = 3. ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003e(G) Schematic of \u003cem\u003ein vivo\u003c/em\u003e LAC administration. Mice were stimulated with LAC at 0 h and 8 h, followed by collection of liver and kidney tissues for detection and analysis.\u003c/p\u003e\n\u003cp\u003e(H) Western blot and RT-PCR analyses of\u003cem\u003e SIRT1\u003c/em\u003e in LAC-stimulated mice. Data are presented as mean ± SD; n = 4. ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003e(I) Western blot analysis and densitometric quantification of the key upstream factor of the JAK-STAT signaling pathway (\u003cem\u003ep-STAT1\u003c/em\u003e) in mice co-treated with GPR132-PKA-SIRT1 pathway inhibitors (H-89, EX527), LAC, and IFN-α. Data are presented as mean ± SD; n = 3. ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e(J) Schematic model summarizing the molecular mechanism by which infection-induced LAC suppresses JAK-STAT antiviral signaling pathway and exacerbates tissue inflammatory damage.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8905265/v1/b0831f678d07fc6261438515.png"},{"id":105904067,"identity":"9a4dcb25-79f5-41f5-8c80-6a43aaeadc6f","added_by":"auto","created_at":"2026-04-01 10:03:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8696408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombined Administration of Stiripentol and IFN-α Synergistically Inhibits Viral Replication and Inflammatory Responses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Intracellular and serum LAC levels in VSV and H1N1 Infected A549 Cells and Mice treated with Stiripentol. Data are presented as mean ± SD; n = 3. ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e(B) Schematic of infected cells treated with Stiripentol combined with IFN-α. Cells were infected with VSV or H1N1, followed by treatment with stiripentol at 12 h post-infection and IFN-α at 24 h post-infection; alternatively, cells were pretreated with IFN-α alone at 2 h prior to infection.\u003c/p\u003e\n\u003cp\u003e(C) Western blot analysis of \u003cem\u003ep-STAT1, STAT1\u003c/em\u003e of the JAK-STAT signaling pathway in infected cells(A549) treated with Stiripentol combined with IFN-α as described in (A).\u003c/p\u003e\n\u003cp\u003e(D) Schematic of infected mice treated with stiripentol combined with IFN-α. Mice were infected with VSV or H1N1, followed by combined therapy with IFN-α and stiripentol at 24 h post-infection, with an additional dose of stiripentol administered at 36 h post-infection; alternatively, mice were treated with IFN-α or stiripentol alone at 24 h or 36 h post-infection. Mouse blood, liver, and lung tissues were collected.\u003c/p\u003e\n\u003cp\u003e(E) Western blot analysis and densitometric quantification of key upstream factors of the JAK-STAT signaling pathway in liver and lung tissues of infected mice treated with Stiripentol combined with IFN-α as in (C). Data are presented as mean ± SD; n = 3. ∗p \u0026lt; 0.05, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e(F) H\u0026amp;E staining and IHC (LY6G) staining of lung tissues from infected mice treated with Stiripentol combined with IFN-α. Histopathological changes were observed; liver and lung tissues were subjected to IHC staining for LY6G.\u003c/p\u003e\n\u003cp\u003e(G) Quantification of LY6G-positive cells in lung tissues. Data are presented as mean ± SD; n = 3. ∗∗∗p \u0026lt; 0.001, ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8905265/v1/00ce7017e8a2fd4becdd940d.png"},{"id":105906334,"identity":"75c67f5d-072f-4057-b2bd-104b63c9c0e1","added_by":"auto","created_at":"2026-04-01 10:20:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":38771799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8905265/v1/19fa3c5a-bdfb-4de0-afb2-3a94e7243c4e.pdf"},{"id":105728091,"identity":"12d987d9-50fa-47c5-b4e3-ce1561aefb01","added_by":"auto","created_at":"2026-03-30 11:09:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17110732,"visible":true,"origin":"","legend":"Host-Derived Lactic Acid Disrupts IFN Clinical Efficacy via Antiviral Inhibition and Proinflammatory Amplification","description":"","filename":"SupplementaryMaterialsfinal1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8905265/v1/8af9dbe04106fd88182a80ca.docx"},{"id":105055504,"identity":"9a038f2f-0770-4ec5-bcc1-8e8858062b0f","added_by":"auto","created_at":"2026-03-20 11:34:33","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5821988,"visible":true,"origin":"","legend":"","description":"","filename":"GraphAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-8905265/v1/0653795034df2f496f118261.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Host-Derived Lactic Acid Disrupts IFN Clinical Efficacy via Antiviral Inhibition and Proinflammatory Amplification","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDriven by accelerating globalization and ongoing ecological alterations, viral infections have emerged as a major disruptive threat to global public health. Frequent cross-species transmission and global viral pandemics not only pose a life-threatening risk to human health, leading to substantial morbidity and mortality, but also trigger systemic public health crises with profound socioeconomic impacts(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Although vaccination and antiviral interventions offer effective approaches to contain known epidemic viruses, their inherent limitations\u0026mdash;narrow viral tropism, susceptibility to escape mutants, and a development cycle that lags behind viral evolution\u0026mdash;render them ineffective against emerging and unknown viral pathogens. Thus, the development of broad-spectrum antiviral agents that are independent of specific viral types and possess pan-inhibitory activity has become a core imperative in global public health. This holds important strategic value for constructing a pre-emptive defense barrier against emerging viral infections and safeguarding global public health security.\u003c/p\u003e \u003cp\u003eWithin the host\u0026rsquo;s immune regulatory network against viral infections, interferons (IFNs) hold a central role as the primary known broad-spectrum antiviral factors (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), exerting potent inhibitory effects on diverse viruses regardless of viral species. Extensive preclinical research studies have demonstrated robust antiviral activity of IFNs in both \u003cem\u003ein vitro\u003c/em\u003e cellular systems and \u003cem\u003ein vivo\u003c/em\u003e animal models. For instance, pretreatment with recombinant IFN-α reduces influenza virus titers by 10\u0026sup2;\u0026ndash;10\u0026sup3; fold in MDCK cells(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), through targeting viral RNA replication and protein translation. Conversely, IFN-α/β receptor-deficient mice succumb to lethal infection within 3\u0026ndash;5 days following low-dose challenge with vesicular stomatitis virus (VSV) or Semliki Forest virus (SFV), with tissue viral loads elevated by 10\u0026sup3;\u0026ndash;10⁴ fold relative to wild-type littermates. In contrast, wild-type mice rapidly induce IFN responses to clear the virus within 72 hours, with 100% survival(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). During the COVID-19 pandemic, IFNs similarly exhibited significant antiviral potential, with multiple \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies confirming their efficacy in suppressing viral replication and mitigating host tissue damage. Collectively, these findings establish IFNs as indispensable effector molecules of innate immunity, thereby providing a theoretical and experimental foundation for the development of IFN-based broad-spectrum antiviral strategies\u0026mdash;addressing the unmet global public health need highlighted earlier.\u003c/p\u003e \u003cp\u003eHowever, a profound discrepancy persists between the potent broad-spectrum antiviral potential of IFNs in preclinical studies and their limited clinical utility(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Currently, their clinical indications are highly restricted\u0026mdash;approved only for a handful of conditions including chronic hepatitis B (CHB), chronic hepatitis C (CHC), and hairy cell leukemia(\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Even for HCV, IFNs are not first-line therapy and are reserved for specific patient cohorts meeting strict eligibility criteria(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).More critically, for acute viral infections with higher incidence and broader public health impact (e.g., acute influenza, COVID-19, dengue fever), IFNs are not recommended for routine use; instead, their application is hampered by suboptimal efficacy(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) and elevated risk of adverse events(\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). For instance, while IFN-α treatment shortens viral shedding by 1\u0026ndash;2 days in acute influenza patients, it fails to substantially alleviate clinical manifestations such as fever and cough, and may induce adverse reactions including headache, fatigue, and myelosuppression(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In early acute COVID-19, despite \u003cem\u003ein vitro\u003c/em\u003e and animal studies demonstrating viral replication inhibition by IFN-α/λ, multicenter randomized controlled trials (RCTs) show no improvement in hospitalization or severe disease progression rates among mild cases. For severe cases, IFN therapy may even exacerbate lung injury via excessive inflammatory activation, worsening clinical outcomes(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).The core regulatory mechanisms underlying this stark divide between IFN's preclinical promise and clinical performance remain incompletely defined, highlighting an urgent need for systematic mechanistic investigations to facilitate the rational clinical deployment of IFNs.\u003c/p\u003e \u003cp\u003eEmerging evidence indicates that the clinical limitations of IFNs stem from multifaceted factors. Animal studies have confirmed that the protective effects of IFNs against infections such as SARS-CoV and MERS-CoV are strictly dependent on prophylactic administration or early post-infection intervention(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). However, due to limitations in viral detection technologies and patient presentation timelines, early clinical intervention is rarely achievable, resulting in missed optimal treatment windows. Mechanistically, IFNs exert antiviral effects primarily by activating the JAK-STAT pathway\u0026mdash;inducing antiviral effector molecule expression to suppress viral replication, while regulating the activation and differentiation of dendritic cells and T cells to constrain excessive inflammation(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). By the time acute viral infection patients seek clinical care, however, viruses have already completed initial replication, immune responses are initiated, and some patients have entered a phase of hyperinflammation. At this stage, IFN therapy not only fails to exert antiviral effects but also its forced activation of the inflammatory responses and worsening disease severity(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Although backed by some clinical studies, the causes of IFN therapeutic failure and adverse effects remain unclear, with no effective countermeasures available. Elucidating the mechanisms underlying IFN therapeutic failure and adverse effects in late-stage treatment is critical for optimizing IFN usage strategies, expanding clinical applications, and developing broad-spectrum antiviral agents, thus offering novel therapeutic directions for addressing current and future emerging viral pandemics.\u003c/p\u003e \u003cp\u003eTo address this key scientific question, we integrated analyses of clinical samples, and cell/animal models of multi-viral infections, demonstrating that host-derived lactic acid (LAC) produced following established viral infection is the central driver of IFN therapeutic failure. Lactate not only directly suppresses IFN antiviral activity but also induces cytokine storms in the presence of IFNs, exerting dual effects that exacerbate host damage and undermine clinical outcomes. Mechanistically, lactic acid directly inhibits IFN antiviral signaling transduction via activating cell membrane receptors and downstream SIRT1 signaling. Concurrently, in synergy with IFNs, lactic acid potently activates the NF-κB pathway to induce inflammatory storms, forming a double negative feedback loop characterized by \"antiviral failure and enhanced inflammation\". Based on this mechanism, we developed a specific combination therapy of IFN plus the lactic acid dehydrogenase inhibitor stiripentol\u0026mdash;an FDA-approved antiepileptic drug. This regimen effectively reduces host lactic acid production, reverses LAC-mediated antiviral suppression and pro-inflammatory effects, and achieves the dual benefit of \"antiviral activity\u0026thinsp;+\u0026thinsp;inflammation control\" with IFN therapy. Successfully overcoming some clinical limitations of IFNs, this combined therapy improves late-stage treatment efficacy and reduces side effects, providing a novel strategy for the development of broad-spectrum antiviral agents and offering new therapeutic prospects for combating current and future viral pandemics.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Interferon treatment loses efficacy and induces a proinflammatory response in the context of established viral infection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a central effector molecule of the innate immune network, interferons (IFNs) are well-recognized key broad-spectrum antiviral agents. Their potent antiviral activity, independent of viral species, has been fully validated both \u003cem\u003ein vitro\u003c/em\u003e cell systems and \u003cem\u003ein vivo\u003c/em\u003e murine models. However, a stark contrast exists between the robust antiviral potential of IFNs in preclinical studies and their severe clinical limitations. Currently, clinical indications of IFNs are extremely narrow\u0026mdash;approved only for a few disorders such as CHB and CHC, and often not as first-line therapy options. For acute viral infections with high incidence and broad impact\u0026mdash;including influenza and COVID-19\u0026mdash;IFNs are not recommended for routine use due to limited efficacy and elevated risk of adverse events. To date, the mechanisms underlying IFN therapeutic failure and adverse effects remain elusive, and no effective solutions exist to address this clinical dilemma, which greatly hindering the full realization of their clinical potential and the development of broad-spectrum antiviral strategies.\u003c/p\u003e\n\u003cp\u003eTo systematically investigate the core mechanisms limiting the clinical utility of IFNs and define the critical time window for IFN-\u0026alpha; intervention, we established multiple virus-infected cell models (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). By varying the timing of treatment and validating across multiple viruses and cell types, we elucidated its mode of action and universality. We selected two representative viruses: one is Influenza virus, a respiratory-transmitted pathogen responsible for multiple global pandemics with substantial mortality. Its seasonal epidemics, high mutation rate, limited vaccine protection, and narrow clinical window for antiviral drugs make it a major public health concern. The other is Vesicular stomatitis virus (VSV), a widely used laboratory model virus with broad tropism and distant phylogenetic relationship to influenza virus, facilitating validation of mechanism\u0026rsquo;s generality. Combined results showed that pretreatment with 2 nM IFN-\u0026alpha; 2 h prior to infection effectively suppressed viral replication, whereas IFN-\u0026alpha; antiviral activity was significantly attenuated once infection was established (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Specifically, pre-infection IFN-\u0026alpha; pretreatment (-2 h) markedly inhibited the replication of influenza A virus (H1N1) and VSV in human non-immune A549 cells, reducing viral RNA and protein expression levels. This effect was consistently validated in immune cells (murine peritoneal macrophages, PM) and other primary cells (murine lung fibroblasts, MLF) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC; Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C). In contrast, IFN-\u0026alpha; administration following viral infection significantly impaired antiviral efficacy with even early post-infection treatment (4 h) resulted in reduced viral activity (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC; Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C). Furthermore, considering the pro-inflammatory potential of IFNs, we investigated this aspect. Results indicated that IFN-\u0026alpha; pretreatment significantly suppressed virus-induced secretion of proinflammatory cytokines (\u003cem\u003eIL-1\u0026beta;, IL-6, TNF-\u0026alpha;\u003c/em\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eD). However, after infection is established, IFN-\u0026alpha; therapy not only lacked anti-inflammatory effects but also significantly exacerbated inflammatory responses at later stages, particularly in influenza virus-infected cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD; Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eD). These findings clearly demonstrate that IFN antiviral efficacy is strictly time-dependent: optimal antiviral effects are only achieved when administered pre-infection. Following established viral infection, therapeutic efficacy is drastically diminished and accompanied by proinflammatory adverse effects\u0026mdash;an observation independent of virus and cell type, with substantial generality.\u003c/p\u003e\n\u003cp\u003eTo further validate the \u003cem\u003ein vivo\u003c/em\u003e relevance of \u003cem\u003ein vitro\u003c/em\u003e findings, we recapitulated the experimental design in murine models (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Mice were infected with different viruses and treated with IFN-\u0026alpha; either pre-infection or post-infection, with systematic analysis of viral replication and pathological damage across infected tissues. Results demonstrated that compared to pre-infection pretreatment, IFN-\u0026alpha; administration following infection significantly reduced antiviral potency. Even treatment initiated one day post-infection showed compromised efficacy, with later treatments correlating with weaker antiviral activity (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF; Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eE and S1F). In contrast, IFN-\u0026alpha; pretreatment effectively suppressed viral replication in mice (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF; Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eE and S1F). More importantly, compared to infection alone, post-infection IFN-\u0026alpha; therapy significantly exacerbated inflammatory responses in the liver and lung tissues of mice, inducing more severe histopathological damage and inflammatory cell infiltration (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG and H; Figure S2A-C). Conversely, IFN-\u0026alpha; pretreatment effectively inhibited viral replication, significantly attenuating inflammatory responses and histopathological damage (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE-H; Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eD-F; Figure S2). This pattern was consistently observed across different viral infection models and tissue types, confirming its \u003cem\u003ein vivo\u003c/em\u003e generality.\u003c/p\u003e\n\u003cp\u003eIn summary, our study demonstrates that IFN-\u0026alpha; administration prior to viral infection fully exerts its antiviral effects while significantly reducing inflammatory responses and virus-induced histopathological damage. In contrast, IFN-\u0026alpha; therapy initiated after viral infection is established not only results in drastically diminished antiviral activity but also exacerbates inflammatory responses and tissue injury. Notably, this phenomenon exhibits broad generality across different viral infection models, cell types, and \u003cem\u003ein vivo\u003c/em\u003e tissues. Fundamentally, the prophylactic intervention models of \u0026quot;pre-infection pretreatment\u0026quot; or \u0026quot;concomitant administration\u0026quot; used in preclinical studies are not clinically feasible, as prophylactic pre-infection use is rarely achievable, and optimal timing is difficult to determine. Clinical interventions for diagnosed patients typically occur after infection is established, where treatment induces a double negative effect of \u0026quot;antiviral failure and enhanced side effects\u0026quot;\u0026mdash;this constitutes the core reason why preclinical IFN efficacy cannot be fully recapitulated in clinical practice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Established Viral Infection Suppresses IFN-\u0026alpha; Signaling Transduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antiviral effects of IFN-\u0026alpha; are primarily mediated by its binding to interferon receptors, leading to activation of the downstream JAK-STAT signaling pathway and subsequent induction the expression of core antiviral factors including MX1(\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e) and OAS1(\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e). In this study, we observed a significant attenuation of IFN-\u0026alpha; antiviral activity following established viral infection. We therefore hypothesized that impaired JAK-STAT signaling transduction following infection is the key mechanism underlying IFN-\u0026alpha; therapeutic failure.\u003c/p\u003e\n\u003cp\u003eTo test this hypothesis, we administered IFN-\u0026alpha; at two critical time points (12 h and 24 h) following established influenza or VSV infection (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), using direct IFN-\u0026alpha; treatment (without viral infection) as a control. Results showed that IFN-\u0026alpha; failed to effectively activate the JAK-STAT signaling pathway following established viral infection in both non-immune cells (A549, MLF) and immune cells (PM). In contrast, IFN-\u0026alpha; treatment efficiently activated this pathway and its downstream effectors in normal, uninfected cells. Specifically, following infection with different viruses, the phosphorylation of STAT1\u0026mdash;a key upstream transducer in the IFN signaling pathway\u0026mdash;was significantly suppressed (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD; Figure S4A). Concurrently, the expression levels of downstream core interferon-stimulated genes (\u003cem\u003eISGs\u003c/em\u003e), including \u003cem\u003eISG15, MX1, IFIT1\u003c/em\u003e, and \u003cem\u003eOAS1\u003c/em\u003e, were markedly downregulated (Figure S3A and S3B; Figure S4B and S4C). These findings demonstrate that impaired JAK-STAT signaling transduction following established viral infection is the core mechanism of IFN-\u0026alpha; antiviral failure. This regulatory effect was consistent across different viruses and cell types, indicating broad generality.\u003c/p\u003e\n\u003cp\u003eTo further validate these findings \u003cem\u003ein vivo\u003c/em\u003e, we established a murine model of H1N1 influenza virus infection. Mice were treated with IFN-\u0026alpha; (200 ng per mouse) post-infection, and lung tissues were collected for analysis (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). \u003cem\u003eIn vivo\u003c/em\u003e results were fully consistent with \u003cem\u003ein vitro\u003c/em\u003e observations: compared to IFN-\u0026alpha; treatment in uninfected mice, IFN-\u0026alpha; treatment following viral infection failed to effectively activate the JAK-STAT signaling pathway in mouse lung tissues. Expression of key downstream effector genes (\u003cem\u003eISG15, MX1, IFIT1\u003c/em\u003e, and \u003cem\u003eOAS1\u003c/em\u003e) was significantly reduced, ultimately limiting IFN-\u0026alpha; antiviral efficacy (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). These \u003cem\u003ein vivo\u003c/em\u003e data confirm that established viral infection specifically suppresses IFN-\u0026alpha;-mediated JAK-STAT signaling transduction and downstream effector expression.\u003c/p\u003e\n\u003cp\u003eIn summary, through integrated \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments, this study demonstrates that IFN-\u0026alpha; fails to effectively activate the JAK-STAT signaling pathway following established viral infection\u0026mdash;this constitutes the key reason for its its diminished antiviral potency. Importantly, this mechanism exhibits generality across different cell types and viral infection models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Lactic Acid Mediates the Suppression of IFN-\u0026alpha; Antiviral Signaling After Viral Infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior studies have confirmed that IFN-\u0026alpha;-mediated JAK-STAT signaling is significantly suppressed following established viral infection, and this inhibitory effect is conserved across multiple cell types and viral infection models. This suggests the existence of a universal regulatory mechanism that transcends infection contexts and cellular backgrounds to impair the antiviral function of the IFN pathway.\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular mechanism underlying this universal suppression, we performed transcriptomic sequencing on VSV infected PM cells. In the present study, an in-depth analysis was conducted on the most significantly upregulated genes following infection. Combined with the focused mining of differentially expressed genes (DEGs) and the results of TOP 20 pathway enrichment analysis, our findings revealed that: after viral infection, in addition to the significant activation of immune-related pathways, glucose metabolism-related pathways were also significantly upregulated, among which the glycolysis pathway-related genes exhibited the most prominent changes in expression, (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-C), indicating that the glycolytic pathway is prominent activated by viruses. To verify the causal link between aberrant glucose metabolism and JAK-STAT pathway suppression, we pretreated A549 and PM cells with key upstream/downstream glycolytic inhibitors (2-deoxy-D-glucose [2-DG], lactate dehydrogenase inhibitor Oxamate, and pyruvate transport inhibitor UK-5099), followed by IFN-\u0026alpha; stimulation during viral infection (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). Results demonstrated that inhibiting early glycolytic steps and lactate dehydrogenase effectively restored IFN-\u0026alpha;-mediated JAK-STAT signaling activation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE-F), indicating that the impaired IFN-\u0026alpha; responsiveness in the late phase of viral infection is closely associated with lactate dehydrogenase, a key downstream glycolytic enzyme.\u003c/p\u003e\n\u003cp\u003eLactate dehydrogenase (LDH) primarily catalyzes the metabolism of pyruvate to lactate, whereas inhibition of LDH restores interferon (IFN) activity (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). Building on this observation, we further investigated the role of lactate in suppressing IFN function. We found that intracellular lactic acid levels increased progressively over time following viral infection, with a significant elevation observed as early as the acute phase of infection (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG)\u0026mdash;consistent with the early impairment of IFN-mediated antiviral activity following infection (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Furthermore, in murine models, serum LAC levels were significantly elevated in virus-infected mice (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH). More importantly, analysis of clinical samples confirmed the pathological relevance of this phenomenon: COVID-19 patients exhibited significantly higher LAC levels relatived to healthy individuals (physiological normal concentration of lactate, 0.5\u0026ndash;1.6 mmol/L) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI, left). Dynamic monitoring of LAC levels in influenza patients revealed a marked upward trend 3 days after positive infection detection, with no significant change at initial positive infection detection (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI, right)\u0026mdash;consistent with the progressive elevation of lactic acid induced by viral infection. Collectively, these results from cellular, animal, and clinical studies consistently demonstrate that viral infection induces a significant increase in host LAC levels, with clear clinical relevance.\u003c/p\u003e\n\u003cp\u003eBased on these observations, we propose a central hypothesis: Lactate derived from glucose metabolism is the key effector inhibiting IFN-\u0026alpha;-mediated JAK-STAT signaling activation during viral infection. Notably, lactate exerts concentration-dependent effects (physiological vs. supraphysiological). Elevated lactate (exceeding physiological levels) was detected in our cellular models, murine specimens, and clinical samples post-infection, with concentrations reaching up to 10 mmol/L in subsets of patients and mice (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG-I). To better reflect clinical practice\u0026mdash;where interferon therapy is typically initiated after infection establishment, when patients already exhibit elevated lactate (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI)\u0026mdash;we used supraphysiological lactate concentrations in our treatment regimens, and interferon treatment was given after lactic acid treatment. A549 cells, PMs, and MLFs were pretreated with 10 mM lactic acid for 24 h, followed by IFN-\u0026alpha; stimulation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI). Results demonstrated that lactic acid directly and significantly suppressed IFN-\u0026alpha;-mediated JAK-STAT signaling activation. Specifically, in the presence of lactic acid, IFN-\u0026alpha;-induced phosphorylation of \u003cem\u003eSTAT1\u003c/em\u003e (a key upstream molecule in the pathway) and the expression of downstream core antiviral genes (\u003cem\u003eISG15\u003c/em\u003e, \u003cem\u003eMX1\u003c/em\u003e, \u003cem\u003eIFIT1\u003c/em\u003e, \u003cem\u003eOAS1\u003c/em\u003e) were significantly inhibited across multiple cell types (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eJ; Figure S5A and S5B). These findings clearly confirm that elevated host lactic acid induced by viral infection directly inhibits IFN signaling transduction, thereby impairing host antiviral capacity.\u003c/p\u003e\n\u003cp\u003eTo verify the \u003cem\u003ein vivo\u003c/em\u003e generality of this mechanism, we reproduced the experiments in murine models (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eK). Results were fully consistent with \u003cem\u003ein vitro\u003c/em\u003e observations: lactic acid significantly suppressed IFN-\u0026alpha;-mediated JAK-STAT signaling activation in mice (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eL; Figure S5C). Compared to direct IFN-\u0026alpha; treatment, the presence of lactic acid reduced STAT1 phosphorylation levels and significantly inhibited the expression of downstream antiviral genes (\u003cem\u003eISG15\u003c/em\u003e, \u003cem\u003eMX1\u003c/em\u003e, \u003cem\u003eIFIT1\u003c/em\u003e, \u003cem\u003eOAS1\u003c/em\u003e) in mouse lung tissues.\u003c/p\u003e\n\u003cp\u003eIn summary, this study demonstrates that established viral infection specifically induces excessive lactic acid production in the host. As a key regulatory molecule, lactic acid directly impairs the antiviral efficacy of IFN-\u0026alpha; by targeting and inhibiting IFN-\u0026alpha;-mediated JAK-STAT signaling activation. This regulatory mechanism is valid across multiple cell types and murine \u003cem\u003ein vivo\u003c/em\u003e models. Furthermore, the consistent elevation of LAC levels in clinical patients with COVID-19, influenza, and other infections indicates that lactic acid is not merely a metabolic byproduct of infection but a key factor contributing to viral immune escape and disease progression\u0026mdash;further highlighting the generality and clinical significance of this mechanism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Lactic Acid Triggers the Proinflammatory Effects of Interferon\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior studies, along with our own data, have established that IFN therapy significantly exacerbates inflammation following established viral infection (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Given that LAC levels are progressively elevated in cells, murine models, and clinical patients over the course of infection (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD-F), and that LAC is a key regulatory factor inhibiting the antiviral activity of IFN-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e; Figure S5), we hypothesized that LAC may contribute to disease progression by amplifying IFN-\u0026alpha;-induced inflammatory responses\u0026mdash;prompting targeted investigations.\u003c/p\u003e\n\u003cp\u003eWe first performed correlation analyses on clinical samples from influenza and COVID-19 patients. LAC levels in patients were significantly positively correlated with inflammatory markers (interleukin-6 [IL-6], high-sensitivity C-reactive protein [CRP]) and tissue damage markers (creatine kinase-MB [CK-MB], lactic acid dehydrogenase [LDH]) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). This clinical association between elevated LAC, inflammation, and tissue injury provided a foundation for mechanistic inquiry. To define the central role of LAC in inflammatory regulation, we further investigated the inflammatory response characteristics of virus-infected cells in the presence of LAC (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Results showed that compared to viral infection alone, LAC treatment significantly promoted the expression of key proinflammatory cytokines including \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e, \u003cem\u003eIL-1\u0026alpha;\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC; Figure S6A-C)\u0026mdash;a phenomenon conserved across different viral infections and multiple cell types.\u003c/p\u003e\n\u003cp\u003eTo validate the \u003cem\u003ein vivo\u003c/em\u003e relevance of these \u003cem\u003ein vitro\u003c/em\u003e findings, we established a murine model of viral infection combined with LAC treatment (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). Results showed that compared to viral infection alone, LAC treatment significantly enhanced viral replication (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE; Figure S7A and S7B)\u0026mdash;consistent with our previous conclusion that \u0026quot;LAC impairs IFN-\u0026alpha; antiviral activity by targeting and inhibiting IFN-\u0026alpha;-mediated JAK-STAT signaling\u0026quot; (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). More critically, LAC treatment significantly exacerbated tissue inflammatory responses and pathological damage in mice compared to viral infection alone (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG; Figure S7C-E). These \u003cem\u003ein vivo\u003c/em\u003e experiments further confirm that LAC not only inhibits IFN-\u0026alpha; antiviral effects but also amplifies its proinflammatory activity, accelerating disease progression.\u003c/p\u003e\n\u003cp\u003eSince the NF-\u0026kappa;B signaling pathway is the core mediator of proinflammatory cytokine production, we further evaluated the regulatory effect of IFN-\u0026alpha; treatment on cellular inflammatory responses in the presence of LAC (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH). Strikingly, LAC significantly enhanced IFN-\u0026alpha;-induced phosphorylation of p65\u0026mdash;a key upstream molecule in the NF-\u0026kappa;B signaling pathway (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI; Figure S8A-D)\u0026mdash;and upregulated the expression of downstream \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e, \u003cem\u003eIL-1\u0026alpha;\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e. In contrast, treatment with LAC or IFN-\u0026alpha; alone failed to significantly activate the NF-\u0026kappa;B pathway or the expression of related proinflammatory cytokines (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI; Figure S8A-D). This reveals that IFN-\u0026alpha; alone is insufficient to drive substantial inflammation, but in the presence of LAC, it acquires potent proinflammatory capacity, even capable of inducing a cytokine storm in the absence of infection.\u003c/p\u003e\n\u003cp\u003eTo further validate these findings, exclude confounding factors such as viruses, and confirm the direct regulatory role of LAC in IFN-\u0026alpha; proinflammatory effects, we established a murine model of LAC combined with IFN-\u0026alpha; treatment in the absence of viral infection (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Results showed that IFN-\u0026alpha; monotherapy had no significant effect on mouse survival and did not induce obvious tissue damage or inflammatory responses (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eK-M). In contrast, compared to the IFN-\u0026alpha; monotherapy group, the LAC\u0026thinsp;+\u0026thinsp;IFN-\u0026alpha; combination group exhibited significantly reduced mouse survival (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eK), with severe inflammatory infiltration and pathological damage in liver tissues (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eL and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eM). This critical evidence further confirms that LAC can directly trigger IFN-\u0026alpha;\u0026apos;s proinflammatory effects, inducing cytokine storms and exacerbating tissue damage even without viral involvement.\u003c/p\u003e\n\u003cp\u003eIn summary, this study clearly demonstrates that LAC is a key regulatory factor underlying IFN-\u0026alpha; therapeutic failure and adverse effects. It exacerbates virus-associated pathological damage and impairs clinical outcomes through a dual mechanism: (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) impairing IFN-\u0026alpha; antiviral activity by inhibiting IFN-\u0026alpha;-mediated JAK-STAT signaling, thereby promoting viral replication; (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) directly triggering IFN-\u0026alpha;\u0026apos;s proinflammatory potential to induce cytokine storms, exacerbating tissue inflammation and pathological damage, ultimately leading to host death\u0026mdash;even in the absence of viral infection. This finding not only reveals the key mechanism underlying adverse effects of IFN-\u0026alpha; in clinical therapy but also suggests that LAC levels may serve as a potential biomarker for evaluating IFN-\u0026alpha; treatment safety. It provides important theoretical basis and clinical reference for optimizing antiviral therapeutic strategies and improving patient prognosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Lactic Acid Attenuates Antiviral JAK-STAT Signaling Through PKA-SIRT1 Axis Activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaving identified lactic acid as a key driver of impaired interferon-\u0026alpha; (IFN-\u0026alpha;) antiviral function and exacerbated tissue inflammatory damage, this study further dissected its underlying molecular mechanism. Given reports that LAC can modulate target protein function via direct lactylation modification, we first investigated whether it modulates JAK-STAT pathway activation by inducing lactylation of signal transducer and activator of transcription 1 (STAT1). A549 and PM cells were treated with gradient concentrations of LAC, and results showed no significant differences in STAT1 protein expression across groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that LAC does not affect STAT1 protein synthesis or stability. Furthermore, HA-tagged STAT1 recombinant plasmid was overexpressed in 293T cells, and co-immunoprecipitation (Co-IP) assays confirmed that STAT1 was not lactylated (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). These findings clearly rule out the possibility that LAC regulates STAT1 function via direct lactylation modification.\u003c/p\u003e\n\u003cp\u003eLAC has also been shown to exert indirect regulatory effects by activating multiple signaling pathways\u0026mdash;for example, initiating the downstream GPR132-PKA pathway via G protein-coupled receptor 132 (GPR132)(\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e), activating the PKA signaling pathway after transmembrane transport mediated by monocarboxylate transporter 1 (MCT1)(\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e), or regulating gene transcription via the histone acetyltransferase p300 as a key substrate for histone lactylation(\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e) (Figure S9A). However, which component of these signaling pathways mediates LAC\u0026apos;s inhibitory effect on the IFN pathway remains unclear. To address this question, A549 and PM cells were pretreated with the GPR132 antagonist Telmisartan, PKA inhibitor H-89, or MCT1 inhibitor AZD3965, followed by co-stimulation with LAC and IFN-\u0026alpha;. Results demonstrated that targeted inhibition of the GPR132-PKA pathway and MCT1 in A549 and PM cells significantly restored STAT1 phosphorylation levels and the expression of \u003cem\u003eISG15\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). This demonstrates that LAC\u0026apos;s inhibition of the IFN pathway is dependent on MCT1-mediated transmembrane transport, GPR132 receptor binding, and downstream PKA signaling activation\u0026mdash;with PKA downstream molecules potentially serving as key regulatory nodes.\u003c/p\u003e\n\u003cp\u003eTo identify the key downstream mediator of PKA, we performed screening of downstream factors. Previous studies have shown that downstream of PKA can regulate biological functions by activating AMPK(\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e) and SIRT1(\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e). Concomitantly, lactate can also directly induce histone lactylation via p300. We pretreated cells and lung with the AMPK inhibitor Compound C, SIRT1 inhibitor EX527, or p300 inhibitor C646, followed by co-stimulation with LAC and IFN-\u0026alpha;. Results showed that only the SIRT1 inhibitor EX527 effectively restored IFN-\u0026alpha;-mediated JAK-STAT pathway activation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE; Figure S9B), while AMPK and p300 inhibitors had no such effect\u0026mdash;confirming that SIRT1 is the key downstream molecule mediating LAC\u0026apos;s inhibition of the IFN pathway. Meanwhile, high-throughput sequencing analysis of bronchoalveolar lavage fluid (BALF) from COVID-19 patients and healthy controls demonstrated a significant upregulation of SIRT1 in the patient cohort compared other SIRT families. (Figure S9C). Previous studies have demonstrated that SIRT1, an NAD+-dependent deacetylase, negatively regulates the JAK-STAT pathway by promoting STAT1 acetylation and inhibiting its phosphorylation(\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e), but its role in LAC-mediated IFN inhibition has not been reported. We found that LAC treatment significantly upregulated SIRT1 protein expression in multiple cell lines including A549 and PM (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). At the \u003cem\u003ein vivo\u003c/em\u003e level, SIRT1 expression was also significantly increased in mouse liver and kidney tissues following intraperitoneal LAC injection (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). Consistent \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e results confirm that LAC specifically induces excessive SIRT1 expression. Lastly, mice were pretreated with the PKA inhibitor H-89 and the SIRT1 inhibitor EX527, followed by co-stimulation with LAC and IFN-\u0026alpha;. Our results demonstrated that lactic acid significantly suppressed the activation of the JAK-STAT signaling pathway in mouse lung tissues, while H-89 and EX527 effectively reversed this lactic acid-mediated inhibitory effect. (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e\n\u003cp\u003eIn summary, this study demonstrates that excessive LAC accumulation induced by viral infection can specifically upregulate SIRT1 expression via PKA activation, inhibiting STAT1 phosphorylation and blocking the JAK-STAT antiviral signaling pathway\u0026mdash;ultimately impairing IFN-\u0026alpha; antiviral efficacy and exacerbating tissue inflammatory damage (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eJ). This mechanism not only reveals a potential strategy for viral escape from host antiviral immunity but also provides a key pathophysiological explanation for the poor antiviral response and inflammation-related adverse effects (e.g., hepatorenal impairment) observed in clinical IFN-\u0026alpha; therapy. Viral infection patients often exhibit abnormally elevated LAC metabolism in clinical settings, and the LAC-SIRT1-JAK-STAT regulatory axis identified in this study suggests that targeting lactic acid metabolism may serve as a potential intervention strategy to improve IFN-\u0026alpha; therapeutic response and reduce treatment-related damage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Combined Administration of Stiripentol and IFN-\u0026alpha; Synergistically Inhibits Viral Replication and Inflammatory Responses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies, including our own, have clearly identified LAC as a key regulatory factor that impairs IFN-\u0026alpha; antiviral efficacy and exacerbates virus-induced tissue inflammatory damage. Based on this, targeted inhibition of lactic acid production may serve as an effective strategy to restore IFN-\u0026alpha; antiviral activity and alleviate inflammation-related damage associated with viral replication.\u003c/p\u003e\n\u003cp\u003eBuilding on these findings, we developed a specific combined therapeutic regimen of IFN-\u0026alpha; plus the lactic acid dehydrogenase (LDH) inhibitor stiripentol. As an FDA-approved clinical drug for the treatment of severe myoclonic epilepsy in infants(\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e), stiripentol\u0026rsquo;s classical mechanism of action involves regulating \u0026gamma;-aminobutyric acid (GABA)-ergic nervous system function to inhibit excessive neuronal excitation(\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e). Critically, it also inhibits the property of inhibiting the activity of LDH\u0026mdash;a key enzyme in lactic acid metabolism\u0026mdash;thereby blocking lactic acid production at its source. Previous studies have confirmed that stiripentol can downregulate the lactylation modification level of NBS1 protein by inhibiting lactic acid production in gastric cancer cells, reduce tumor cell DNA repair capacity, and thereby overcome tumor radioresistance and chemoresistance(\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eStiripentol was verified to effectively suppress lactic acid production induced by viral infection in cell and mice(Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequently, we treated it with stetamentanol as shown in the figure (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). Results showed that compared to pre-infection IFN-\u0026alpha; monotherapy, IFN-\u0026alpha; alone almost lost its antiviral activity after infection was established. In contrast, stiripentol treatment effectively rescued IFN-\u0026alpha;-mediated JAK-STAT signaling activation post-infection and significantly inhibited viral replication (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC)\u0026mdash;confirming the core role of the combined therapy in reversing IFN-\u0026alpha; antiviral failure.\u003c/p\u003e\n\u003cp\u003eTo evaluate the \u003cem\u003ein vivo\u003c/em\u003e potential of the combined therapy, we established a murine viral infection model. After infection was established, mice were treated with IFN-\u0026alpha; monotherapy, stiripentol monotherapy, or their combination (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). Results confirmed that post-infection, IFN-\u0026alpha; monotherapy failed to effectively activate the JAK-STAT signaling pathway or induce antiviral gene expression (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE, S10A-B). Stiripentol monotherapy partially restored IFN-\u0026alpha; antiviral function and upregulated the expression of antiviral factors (Figure S10A and S10B). Strikingly, the IFN-\u0026alpha;\u0026thinsp;+\u0026thinsp;stiripentol combination group exhibited the optimal effect, even when administered post- infection, it still drove IFN-\u0026alpha; to efficiently activate the JAK-STAT antiviral signaling pathway, significantly induce antiviral factor expression, and inhibit viral replication (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE; Figure S10A and S10B)\u0026mdash;fully validating the \u003cem\u003ein vivo\u003c/em\u003e antiviral efficacy of the combined therapy.\u003c/p\u003e\n\u003cp\u003eGiven previous evidence that LAC can directly trigger IFN-\u0026alpha;\u0026rsquo;s proinflammatory effects, inducing cytokine storms and exacerbating tissue damage even in the absence of virus, we further evaluated the regulatory role of the combined therapy on post-infection inflammatory responses. Results showed that compared to the high levels of inflammatory cytokines in the IFN-\u0026alpha; monotherapy group, stiripentol monotherapy partially downregulated inflammatory cytokine expression. In contrast, the combination therapy group significantly suppressed infection-induced cytokine storms (Figure S11A and S11B), demonstrating potent anti-inflammatory activity. Histopathological analysis further revealed that compared to viral infection alone, IFN-\u0026alpha; monotherapy post-infection significantly increased inflammatory neutrophil infiltration in mouse liver and lung tissues, inducing more severe histopathological damage (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG; Figure S11C-E). Stiripentol monotherapy had no significant improvement on tissue damage or inflammatory neutrophil infiltration. However, compared to the infection-only group and each monotherapy group, the combination therapy group showed significantly reduced inflammatory neutrophil infiltration and markedly alleviated histopathological damage in liver and lung tissues (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG; Figure S11C-E)\u0026mdash;achieving synergistic benefits of \u0026quot;antiviral activity\u0026thinsp;+\u0026thinsp;inflammation control\u0026quot;.\u003c/p\u003e\n\u003cp\u003eA core bottleneck in current clinical application of IFN-\u0026alpha; lies in the impracticality of prophylactic intervention strategies widely used in preclinical studies (e.g., \u0026quot;pre-infection pretreatment\u0026quot; or \u0026quot;concomitant administration\u0026quot;). Prophylactic administration prior to infection cannot be routinely performed clinically, nor can the optimal timing of administration be accurately determined. Clinical therapeutic interventions for confirmed patients mostly initiate after infection is established, where IFN-\u0026alpha; monotherapy often leads to a dualseed dilemma of \u0026quot;antiviral failure\u0026thinsp;+\u0026thinsp;enhanced inflammatory side effects\u0026quot;. The core advantage of this combined therapy is its significant efficacy even when intervention is initiated post-infection\u0026mdash;completely breaking the strict restriction of traditional IFN-\u0026alpha; therapy on administration timing. It effectively reverses LAC-mediated IFN-\u0026alpha; antiviral inhibition and proinflammatory adverse effects, overcomes some core limitations of clinical IFN-\u0026alpha; application, and significantly improves late-stage infection treatment efficacy while reducing the side effects of monotherapy. This not only provides a novel strategy for the development of broad-spectrum antiviral drugs but also offers new therapeutic options and directions for addressing current and future viral pandemics\u0026mdash;presenting a practical solution to the long-standing problem of limited clinical application of IFN-\u0026alpha; in antiviral therapy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe global spread of emerging and re-emerging viral pandemics continues to pose a severe threat to public health security. While vaccines and traditional antiviral drugs have provided partial solutions for combating known viruses, their inherent limitations have become increasingly prominent: vaccines require a long development cycle, are vulnerable to viral mutation and escape, and can only target specific viral subtypes; traditional antiviral drugs mostly target specific viral proteins, facing challenges such as rapid development of drug resistance and ineffectiveness against unknown viruses. These shortcomings collectively highlight the urgency and strategic significance of developing broad-spectrum antiviral agents that are relatively independent of viral types.\u003c/p\u003e \u003cp\u003eSince the first discovery of interferons (IFNs) by Isaacs and Lindenmann in 1957(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), their pivotal role as core effector molecules of the host innate antiviral immunity has been fully validated through decades of research, making them a central target for studies on antiviral immune mechanisms and clinical translation. In contrast to their prominent role in preclinical research, the current clinical application of IFNs is extremely limited. They are only approved for a few diseases such as chronic hepatitis B and chronic hepatitis C, and are rarely the first-line treatment of choice. For acute viral infections with high incidence and wide impact, such as acute influenza and COVID-19, IFNs are not recommended for routine use; instead, they are strictly restricted due to limited efficacy and high risk of adverse reactions. To date, the core mechanisms underlying IFN clinical failure and side effects remain unclear, and there is a lack of effective solutions to address this clinical dilemma, which has greatly hindered the full realization of their clinical value and the development of broad-spectrum antiviral strategies.\u003c/p\u003e \u003cp\u003eThrough systematic validation using clinical samples, multi-viral infection cell models, and murine models, this study found that the prophylactic intervention modes such as \"pre-infection pretreatment\" or \"concomitant administration\" widely used in preclinical research are rarely feasible in clinical practice. Prophylactic use before infection is difficult to implement, and the optimal timing of administration is even more challenging to determine. Clinical therapeutic interventions for confirmed patients mostly occur at the established infection stage, where drug administration results in a dual negative effect of \"antiviral failure\u0026thinsp;+\u0026thinsp;enhanced side effects\"\u0026mdash;this is the core reason why the basic efficacy of IFNs cannot be translated into clinical outcomes. Mechanistically, this study identified host-derived lactic acid induced by viral infection as the key driver of the clinical bottleneck of IFN therapy, and clarified its dual regulatory mechanism of \"LAC-SIRT1-JAK-STAT/LAC-NF-κB\". Ultimately, a transformative combination therapy of IFN plus stiripentol was developed, providing a novel strategy for broad-spectrum antiviral treatment.\u003c/p\u003e \u003cp\u003eThis study confirmed that IFN therapeutic efficacy is highly dependent on the timing of infection, which provides a key entry point for explaining the discrepancy between its preclinical and clinical efficacy. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrated that IFN pretreatment can effectively inhibit the replication of various viruses and reduce inflammatory responses in multiple cell types; however, administration after the establishment of viral infection not only significantly impairs antiviral activity but also exacerbates cytokine storms and tissue damage. \u003cem\u003eIn vivo\u003c/em\u003e experiments further verified the universality of this rule, suggesting that clinical therapeutic interventions for confirmed patients after established infection are the core cause of poor IFN efficacy. In-depth studies revealed that virus-induced LAC at concentrations far exceeding the normal physiological level serves as the central factor mediating the abnormal function of IFN. Our experimental model was established to simulate clinical infection scenarios. Particularly, with respect to the lactate stimulation intervention step, we designed the treatment sequence based on the clinical pathological process in which lactate is first induced upon infection prior to interferon administration. Subsequent experimental validation confirmed that interferon alone failed to trigger an inflammatory response; instead, lactate produced during viral infection acts as the critical synergistic factor mediating the induction of inflammatory reactions. Transcriptome analysis and metabolic detection confirmed that different viral infections universally activate the host glycolytic pathway, leading to a significant increase in LAC levels in cells and serum\u0026mdash;this phenomenon was validated in clinical samples from COVID-19 and influenza patients, highlighting its pathological relevance. This further indicates that LAC, previously considered a mere metabolic byproduct, may play a key role in infection-related disease progression. Mechanistic experiments clarified that LAC impairs IFN therapeutic effects through a dual mechanism: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) LAC induces excessive SIRT1 expression via the GPR132-PKA signaling pathway, thereby inhibiting STAT1 phosphorylation, blocking the activation of the JAK-STAT antiviral signaling pathway, and directly impairing the viral inhibitory capacity of IFNs; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) LAC synergizes with IFNs to efficiently activate the NF-κB pathway, inducing storms of proinflammatory cytokines such as IL-1β and IL-6, and exacerbating tissue damage. Notably, combined treatment with LAC and IFNs can induce inflammatory damage and mouse death even in the absence of viral infection, confirming that this proinflammatory effect is independent of viral replication and serves as a key inducer of IFN clinical side effects. This mechanism not only clarifies the critical role of LAC in regulating IFN antiviral function but also reveals the central role of SIRT1 in LAC-mediated IFN signaling inhibition, providing a clear molecular target for targeted intervention.\u003c/p\u003e \u003cp\u003eBased on the above mechanisms, this study innovatively developed a \"IFN combined with lactic acid dehydrogenase (LDH) inhibitor stiripentol\" combination therapy, realizing a complete chain of \"mechanism-target-drug\". As an FDA-approved clinical drug, stiripentol's property of inhibiting LDH activity and blocking LAC production was first applied in antiviral therapy. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments confirmed that even when intervention is initiated after the establishment of viral infection, this combination therapy can effectively reduce LAC levels, restore IFN-mediated JAK-STAT pathway activation, and significantly inhibit viral replication; meanwhile, it can potently block LAC-IFN synergistically induced cytokine storms, alleviate hepatopulmonary pathological damage, and achieve dual benefits of \"efficient antiviral activity\u0026thinsp;+\u0026thinsp;precise inflammation control\". The core advantage of this regimen lies in breaking the strict restriction of IFN on administration timing, solving the practical clinical problem of \"difficulty in early intervention\". Additionally, the clinical application basis of stiripentol reduces translational risks, enabling the rapid advancement of clinical research. The aforementioned combination therapy provides novel targets and technical strategies for the development of broad-spectrum antiviral agents. Beyond its substantial antiviral activity, IFN also exerts potent effects in contexts such as antitumor therapy and antibacterial infection. Previous studies have demonstrated that lactic acid is similarly highly expressed in the tumor microenvironment and infected lesions(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), indicating that lactic acid may also interact with interferons. Based on this observation, the relevant mechanism of action and combined therapeutic regimen proposed herein are also expected to open new avenues and provide innovative strategies for the treatment of tumors and other diseases. This offers novel therapeutic approaches and drug options for addressing existing and future unknown diseases as well as viral pandemics, holding significant clinical translational value and public health implications.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eComprehensive information regarding the reagents, consumables, antibodies, and other relevant materials utilized in the experiments is available in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003ePatient samples\u003c/h3\u003e\n\u003cp\u003eAll clinical samples were collected from the Shiyan People's Hospital, Hubei Medical University. Lactate levels, inflammatory factor levels, and myocardial injury markers were collected 210 COVID-19 patients, 23 influenza patients (on Day 1 and Day 3 of illness respectively), and 58 healthy controls from Shiyan People's Hospital, who were admitted between January 2022 and December 2023. All samples were collected with the patients' informed consent and signed written consent forms. The experiment was approved by the Ethics Committee of Shiyan People's Hospital Affiliated to Hubei University of Medicine, and complied with the ethical standards established in the Declaration of Helsinki.\u003c/p\u003e\n\u003ch3\u003eCells\u003c/h3\u003e\n\u003cp\u003eIn this study, various cell lines, including HEK293T, VERO, A549 and MDCK were obtained from the American Type Culture Collection (ATCC). Primary peritoneal macrophages were isolated from mice 5 days following intraperitoneal injection of thioglycollate broth medium. Lung fibroblasts were isolated by mincing mouse lungs and digesting with Hank's solution. These were cultured in DMEM media. All media were supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin. The cells were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO2. It\u0026rsquo;s noteworthy that all cell lines used in the study were regularly tested and confirmed to be free from mycoplasma contamination.\u003c/p\u003e\n\u003ch3\u003eViruses\u003c/h3\u003e\n\u003cp\u003eInfluenza H1N1-PR stocks were propagated in Serum Free Medium for MDCK Cells. VSV-GFP propagated in Serum Free Medium for VERO Cells.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAll mice were maintained in a specific-pathogen-free facility with a 12-hour light/dark cycle and were allowed ad libitum access to food and water. Six-week-old male C57BL/6J wild-type mice were purchased from Changsheng Biotechnology (Liaoning, China). Mice were infected with H1N1 virus via intranasal instillation and with VSV virus via intraperitoneal injection. Additionally, mice were administered IFN-α, LAC and stiripentol via intraperitoneal injection for stimulation. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Hubei University of Medicine.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInfection and transfection\u003c/h3\u003e\n\u003cp\u003eFor infection, A549, PM and MLF cells were infected with DMEM containing viruses with a multiplicity of infection (MOI). For transfection, plasmids were transfected by using Lipofectamine 3000 according to the manufacturers instructions, cells were harvested or further treatment as indicated.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting (IB)\u003c/h3\u003e\n\u003cp\u003eCells were harvested and lysed with lysis buffer (150 mM NaCl, 50 mM Tris-HCl [PH 7.4], 1% Triton X-100, 1mM EDTA [pH 8.0], 0.1% SDS supplemented with a protease inhibitor cocktail) for 30 min at 4\u0026deg;C. The supernatants were collected by centrifugation at 12000 g for 25 min at 4\u0026deg;C. Protein concentrations were determined using the Bradford method. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% non-fat milk dissolved in TBST (phosphate-buffered saline with 0.1% Tween 20), it was incubated with the primary Abs, followed by horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG. The proteins were detected on a BIO-RAD ChemiDoc Imaging System by using Enhanced chemiluminescence (ECL).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation(IP)\u003c/h2\u003e \u003cp\u003eFor protein-protein interactions assays, cells were lysed in RIPA lysis buffer (150 mM NaCl, 0.5% NP-40, 5 mM EDTA) containing a mixture of protease inhibitor (Roche). Primary antibodies were incubated with Protein A/G agarose beads for 30 min at room temperature, followed by incubation with cell lysates for 3 hours with rotation at room temperature (RT). The beads were washed four times with lysis buffer and analyzed by IB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry(IHC) analysis\u003c/h2\u003e \u003cp\u003eThin sections of hepatic and pulmonary tissues were treated to remove paraffin and restore hydration. They were then placed in a sodium citrate buffer with a pH of 6.0 for antigen retrieval and allowed to cool. Following this, endogenous peroxidase activity was suppressed for a duration of 10 min through the application of a 0.3% (v/v) hydrogen peroxide solution in methanol. After being incubated with a 10% goat serum blocking solution for 30 min, the samples were subjected to Ly-6G (1:200) antibodies overnight. Following two rounds of washing, the samples underwent a 30 min incubation at room temperature with a secondary antibody diluted at a ratio of 1:100. The cell nuclei were subsequently stained with hematoxylin following the color development using the DAB kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative RT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells or tissues that have been processed with Trizol. 0.6 \u0026micro;g of total RNA was reversed transcribed to synthesize cDNA by HiScipt Ⅱ Q RT SuperMix, using ChamQ SYBR qPCR Master Mix for qPCR. Real-time quantitative PCR was run in duplicate using iTaq SYBRGreen (Bio-Rad), following manufacturer\u0026rsquo;s instructions. Data are presented as relative mRNA abundance normalized to GAPDH expression in each sample.( Primer sequence information is shown in Table S2)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA-sequencing and data processing\u003c/h2\u003e \u003cp\u003eVirus-infected cells were immediately transferred into 1 ml Trizol, snap-frozen in liquid nitrogen, and kept at \u0026minus;\u0026thinsp;80\u0026deg;C. RNA extraction was performed by GeneDenovo (QIAGEN, China). RNA integrity number (RIN) was determined using the Agilent RNA 6000 Nano Kit for quality control and all samples were above 8.5. After sequencing, samples were aligned to the Ensembl_release111 Genome. For the pathway analysis, GSEA (4.2.3) was used to identify pathway enrichment among the total genes expressed in the PM cells infected with VSVG or mock.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLactate measurements\u003c/h2\u003e \u003cp\u003e L-lactic acid levels in cells and mouse tissues were measured using the L-lactic acid assay kit according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay\u003c/h2\u003e \u003cp\u003ePurified proteins were immobilized onto 96-well plates in a carbonate buffer (200 ng per well) and incubated at 4\u0026deg;C overnight. After five times washing with PBST (PBS with 0.05% Tween 20), blocking was performed using 1% BSA in PBST. Subsequently, Mouse serum samples were added and incubated at room temperature for 2 h. Following five times washing, Secondary antibodies were added for 1 h. Then, the HRP diluted in PBST with 1% BSA was added and incubated for an additional hour. After five times washing, the TMB substrate was applied, and the reaction was stopped by adding 1 M H2SO4. Absorbance at 450 nm was measured and recorded. The obtained data were analyzed using GraphPad Prism 9.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E staining of liver and lung tissues\u003c/h2\u003e \u003cp\u003eHepatic and pulmonary tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4-\u0026micro;m slices. Sections were deparaffinized in xylene, hydrated through a graded ethanol series, stained with filtered Harris hematoxylin for 8 min and 0.5% eosin Y for 3 min, followed by dehydration and clearing. Stained sections were mounted with neutral balsam and visualized under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations from at least three independent experiments. Comparisons of two groups were performed by using two-tailed unpaired Student\u0026rsquo;s t test. Comparisons of multiple groups were performed by using one-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s multiple-comparisons test unless otherwise indicated. Statistical analysis was performed using the GraphPad Prism 7 software package. For all analyses, a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.L., and Z.L. contributed to the study concept and design. M.T., S.C. and J.H. carried out most of the experiments. X.W., M.L., X.L., Z.C., X.X., S.L, M.L., C.H., N.W., Z.Z., and W.D. analysed the data with help from K.L. and Z.L. contributed to the drafting of the manuscript.\u0026nbsp;Q.T., contributed to the reagents.\u0026nbsp;W.D., J.W., and Z.L.\u0026nbsp;contributed to edit the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by research grants from National Natural Science Foundation of China (32188101, 32400131), Hubei Provincial Natural Science Foundation (2023BCB058, JCZRLH202600075), Natural Science Foundation of Hubei Provincial Department of Education (D20242105), China Postdoctoral Science Foundation (2024T170687, 2024M752482, GZB20230541) and the Open Research Fund of State Key Laboratory of Virology and Biosafety (SKLVKF2025014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments involving mice were performed according to the institutional animal ethics committee protocol.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDel Rio C, Collins L F, Malani P. Long-term health consequences of covid-19 [J]. Jama, 2020, 324(17): 1723-1724.\u003c/li\u003e\n\u003cli\u003eAl-Aly Z, Davis H, McCorkell L, et al. Long covid science, research and policy [J]. Nat Med, 2024, 30(8): 2148-2164.\u003c/li\u003e\n\u003cli\u003ePestka S. The interferons: 50 years after their discovery, there is much more to learn [J]. J Biol Chem, 2007, 282(28): 20047-20051.\u003c/li\u003e\n\u003cli\u003eMcNab F, Mayer-Barber K, Sher A, et al. Type i interferons in infectious disease [J]. Nat Rev Immunol, 2015, 15(2): 87-103.\u003c/li\u003e\n\u003cli\u003eLi F, Wang Q J, Zhu B L, et al. Antiviral effects of rhifn-alpha 1 against seven influenza viruses [J]. Zhongguo Yao Li Xue Bao, 1999, 20(8): 709-714.\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller U, Steinhoff U, Reis L F, et al. Functional role of type i and type ii interferons in antiviral defense [J]. Science, 1994, 264(5167): 1918-1921.\u003c/li\u003e\n\u003cli\u003ePan H, Peto R, Henao-Restrepo A M, et al. Repurposed antiviral drugs for covid-19 - interim who solidarity trial results [J]. N Engl J Med, 2021, 384(6): 497-511.\u003c/li\u003e\n\u003cli\u003eAsselah T, Lada O, Moucari R, et al. Interferon therapy for chronic hepatitis b [J]. Clin Liver Dis, 2007, 11(4): 839-849, viii.\u003c/li\u003e\n\u003cli\u003eShapiro S, Gershtein V, Elias N, et al. Mrna cytokine profile in peripheral blood cells from chronic hepatitis c virus (hcv)-infected patients: Effects of interferon-alpha (ifn-alpha) treatment [J]. Clin Exp Immunol, 1998, 114(1): 55-60.\u003c/li\u003e\n\u003cli\u003eWaldmann T A. Cytokines in cancer immunotherapy [J]. Cold Spring Harb Perspect Biol, 2018, 10(12).\u003c/li\u003e\n\u003cli\u003eLocke M C, Fox L E, Dunlap B F, et al. Interferon alpha, but not interferon beta, acts early to control chronic chikungunya virus pathogenesis [J]. J Virol, 2022, 96(1): e0114321.\u003c/li\u003e\n\u003cli\u003eCooke G S, Hung L M, Flower B, et al. Treatment options to support the elimination of hepatitis c: An open-label, factorial, randomised controlled non-inferiority trial [J]. Lancet, 2025, 405(10491): 1769-1780.\u003c/li\u003e\n\u003cli\u003eFinter N B, Chapman S, Dowd P, et al. The use of interferon-alpha in virus infections [J]. Drugs, 1991, 42(5): 749-765.\u003c/li\u003e\n\u003cli\u003eTannock G A, Gillett S M, Gillett R S, et al. A study of intranasally administered interferon a (rifn-alpha 2a) for the seasonal prophylaxis of natural viral infections of the upper respiratory tract in healthy volunteers [J]. Epidemiol Infect, 1988, 101(3): 611-621.\u003c/li\u003e\n\u003cli\u003eGalbraith M D, Kinning K T, Sullivan K D, et al. Specialized interferon action in covid-19 [J]. Proc Natl Acad Sci U S A, 2022, 119(11).\u003c/li\u003e\n\u003cli\u003eHayden F G, Albrecht J K, Kaiser D L, et al. Prevention of natural colds by contact prophylaxis with intranasal alpha 2-interferon [J]. N Engl J Med, 1986, 314(2): 71-75.\u003c/li\u003e\n\u003cli\u003eDavidson S, McCabe T M, Crotta S, et al. Ifn\u0026lambda; is a potent anti-influenza therapeutic without the inflammatory side effects of ifn\u0026alpha; treatment [J]. EMBO Mol Med, 2016, 8(9): 1099-1112.\u003c/li\u003e\n\u003cli\u003eKalil A C, Mehta A K, Patterson T F, et al. Efficacy of interferon beta-1a plus remdesivir compared with remdesivir alone in hospitalised adults with covid-19: A double-bind, randomised, placebo-controlled, phase 3 trial [J]. Lancet Respir Med, 2021, 9(12): 1365-1376.\u003c/li\u003e\n\u003cli\u003eWong L R, Perlman S. Immune dysregulation and immunopathology induced by sars-cov-2 and related coronaviruses - are we our own worst enemy? [J]. Nat Rev Immunol, 2022, 22(1): 47-56.\u003c/li\u003e\n\u003cli\u003eRandall R E, Goodbourn S. Interferons and viruses: An interplay between induction, signalling, antiviral responses and virus countermeasures [J]. J Gen Virol, 2008, 89(Pt 1): 1-47.\u003c/li\u003e\n\u003cli\u003eKim Y M, Shin E C. Type i and iii interferon responses in sars-cov-2 infection [J]. Exp Mol Med, 2021, 53(5): 750-760.\u003c/li\u003e\n\u003cli\u003eXiao H, Killip M J, Staeheli P, et al. The human interferon-induced mxa protein inhibits early stages of influenza a virus infection by retaining the incoming viral genome in the cytoplasm [J]. J Virol, 2013, 87(23): 13053-13058.\u003c/li\u003e\n\u003cli\u003eKristiansen H, Scherer C A, McVean M, et al. Extracellular 2\u0026apos;-5\u0026apos; oligoadenylate synthetase stimulates rnase l-independent antiviral activity: A novel mechanism of virus-induced innate immunity [J]. J Virol, 2010, 84(22): 11898-11904.\u003c/li\u003e\n\u003cli\u003eLiu W, Zhang S, Li Q, et al. Lactate modulates iron metabolism by binding soluble adenylyl cyclase [J]. Cell Metab, 2023, 35(9): 1597-1612.e1596.\u003c/li\u003e\n\u003cli\u003eNgai D, Schilperoort M, Tabas I. Efferocytosis-induced lactate enables the proliferation of pro-resolving macrophages to mediate tissue repair [J]. Nat Metab, 2023, 5(12): 2206-2219.\u003c/li\u003e\n\u003cli\u003eCant\u0026oacute; C, Gerhart-Hines Z, Feige J N, et al. Ampk regulates energy expenditure by modulating nad+ metabolism and sirt1 activity [J]. Nature, 2009, 458(7241): 1056-1060.\u003c/li\u003e\n\u003cli\u003eHahn E L, Halestrap A P, Gamelli R L. Expression of the lactate transporter mct1 in macrophages [J]. Shock, 2000, 13(4): 253-260.\u003c/li\u003e\n\u003cli\u003eZhang D, Tang Z, Huang H, et al. Metabolic regulation of gene expression by histone lactylation [J]. Nature, 2019, 574(7779): 575-580.\u003c/li\u003e\n\u003cli\u003eMedina E A, Oberheu K, Polusani S R, et al. Pka/ampk signaling in relation to adiponectin\u0026apos;s antiproliferative effect on multiple myeloma cells [J]. Leukemia, 2014, 28(10): 2080-2089.\u003c/li\u003e\n\u003cli\u003eGerhart-Hines Z, Dominy J E, Jr., Bl\u0026auml;ttler S M, et al. The camp/pka pathway rapidly activates sirt1 to promote fatty acid oxidation independently of changes in nad(+) [J]. Mol Cell, 2011, 44(6): 851-863.\u003c/li\u003e\n\u003cli\u003eYu S S, Tang R C, Zhang A, et al. Deacetylase sirtuin 1 mitigates type i ifn- and type ii ifn-induced signaling and antiviral immunity [J]. J Virol, 2024, 98(3): e0008824.\u003c/li\u003e\n\u003cli\u003eChiron C, Marchand M C, Tran A, et al. Stiripentol in severe myoclonic epilepsy in infancy: A randomised placebo-controlled syndrome-dedicated trial. Sticlo study group [J]. Lancet, 2000, 356(9242): 1638-1642.\u003c/li\u003e\n\u003cli\u003eFisher J L. The effects of stiripentol on gaba(a) receptors [J]. Epilepsia, 2011, 52 Suppl 2(0 2): 76-78.\u003c/li\u003e\n\u003cli\u003eFisher J L. The anti-convulsant stiripentol acts directly on the gaba(a) receptor as a positive allosteric modulator [J]. Neuropharmacology, 2009, 56(1): 190-197.\u003c/li\u003e\n\u003cli\u003eChen H, Li Y, Li H, et al. Nbs1 lactylation is required for efficient DNA repair and chemotherapy resistance [J]. Nature, 2024, 631(8021): 663-669.\u003c/li\u003e\n\u003cli\u003eIsaacs A, Lindenmann J. Virus interference. I. The interferon [J]. Proc R Soc Lond B Biol Sci, 1957, 147(927): 258-267.\u003c/li\u003e\n\u003cli\u003eLi J, Li Z, Zhang X, et al. Histone lactylation bridges metabolic reprogramming with chromatin-immune crosstalk in triple-negative breast cancer [J]. Cancer Lett, 2025, 639: 218227.\u003c/li\u003e\n\u003cli\u003ePeng X, He Z, Yuan D, et al. Lactic acid: The culprit behind the immunosuppressive microenvironment in hepatocellular carcinoma [J]. Biochim Biophys Acta Rev Cancer, 2024, 1879(5): 189164.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lactic Acid, Type I interferons, Antiviral Activity, Proinflammatory Effects","lastPublishedDoi":"10.21203/rs.3.rs-8905265/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8905265/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEmergent, sudden-onset, and highly prevalent viral pathogens severely threaten public health. Current vaccines/antiviral drugs are limited by narrow tropism, susceptibility to escape mutations, and prolonged development, highlighting an urgent need for broad-spectrum agents. Type I interferons exhibit potent broad-spectrum antiviral activity in preclinical studies but suffer from critical clinical limitations: narrow intervention windows for acute infections, suboptimal efficacy, and notable adverse reactions. The discrepancy between their robust preclinical and limited clinical performance remains mechanistically unclear. Herein, integrating clinical samples and multi-level infection models, we demonstrate that IFNs exert antiviral effects only when administered pre-infection. Once infection is established, IFNs are ineffective yet induce prominent adverse effects, with host-derived lactic acid (LAC) as the key mediator: it promotes viral immune evasion, impairs IFN therapeutic efficacy, triggers inflammatory storms, and elicits adverse reactions. Mechanistically, LAC suppresses IFN activity via membrane receptor-mediated SIRT1 upregulation and synergizes with IFNs to hyperactivate NF-κB, initiating cytokine storms and forming an \"antiviral failure-inflammatory amplification\" feedback loop. Based on this mechanism, we developed a combinatorial therapy of IFNs plus an FDA-approved lactate dehydrogenase inhibitor. This regimen reverses LAC-mediated IFN suppression, mitigates inflammation, and achieves dual \"antiviral + anti-inflammatory\" benefits. Notably, it retains robust efficacy even in late-stage infections, overcoming IFN monotherapy drawbacks and addressing the core bottleneck restricting IFN clinical application. Our study identifies LAC as a pivotal target for broad-spectrum antiviral development and provides a potential strategy to combat emerging viral pandemics.\u003c/p\u003e","manuscriptTitle":"Host-Derived Lactic Acid Disrupts IFN Clinical Efficacy via Antiviral Inhibition and Proinflammatory Amplification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-20 11:34:25","doi":"10.21203/rs.3.rs-8905265/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8e5f54a0-7ff9-41bb-a8c0-5ce9b60f3049","owner":[],"postedDate":"March 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64754415,"name":"Health sciences/Pathogenesis/Infection"},{"id":64754416,"name":"Biological sciences/Microbiology/Virology/Virus\u0026#x2013;host interactions"}],"tags":[],"updatedAt":"2026-04-01T20:15:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-20 11:34:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8905265","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8905265","identity":"rs-8905265","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}